Cat kidney disease marker

ABSTRACT

In view of the increase in the number of cases with nephropathy in aged animals of the cat family, the invention provides a marker for early diagnosis of feline nephropathy which can substitute for the conventional complicated methods of diagnosing nephropathy such as blood examination, kidney radiography using a contrast medium, and ultrasonic image analysis. It was found that cats with nephropathy show markedly lowered levels of excretion of a novel protein (named cauxin) with a molecular weight of 70 K, which is excreted into urine in high concentrations in normal adult cats showing no clinical abnormality. When the protein is used as a marker in diagnosing feline nephropathy, feline nephropathy can be detected in a simple and exact manner.

TECHNICAL FIELD

The present invention relates to a newly identified polypeptide (orprotein) and a polynucleotide (or nucleic acid) (or a part thereof),inclusive of salts thereof, which are useful as markers for diagnosingfeline nephropathy; mutants and derivatives of the polypeptide (orprotein) and of the polynucleotide (or nucleic acid); methods ofproducing the polypeptide (or protein) and polynucleotide (or nucleicacid), or mutants or derivatives thereof; an antibody to the polypeptide(or protein) (or a part thereof), an immunological assay method usingthe antibody, and a reagent to be used therefor; and the use (inclusiveof the use as a diagnostic reagent, or in a diagnostic method, forinstance) of those in diagnosis of feline nephropathy. The presentinvention also relates to a diagnostic method which comprises making adiagnosis of nephropathy frequently encountered in family cats byassaying a urinary protein (cauxin).

BACKGROUND ART

Under considerable daily stresses of life in these days, thoseindividuals who wish to live with a pet are increasing in number, andthe number of pet dogs and cats is increasing year after year. On theother hand, with the advancement in veterinary medicine andpopularization of pet foods, the number of dogs and cats of advanced ageis increasing and, like in humans, the incidence of adult diseases,obesity and nephropathy (renal diseases), among others, is increasing,raising a serious problem. As regards nephropathy, in particular, it isvery difficult to recognize a disease signal at an early stage, assuggested by the saying that the kidney is a silent organ. Therefore,upon veterinary examination in view of some or other symptoms, the renalfunction is found to be in severe disorder in many instances.

In clinical veterinary medicine, the cases of renal diseases have beenincreasing in recent years with the aging of animals of the familyFelidae and, at present, nephropathy is the primary cause of death amongfamily cats. Further, the symptoms of nephropathy are noticed in mostcases only when they are already in advanced stages and, thus, thedisease is often already in an incurable stage at the time ofconsultation with a veterinarian. Thus, chronic renal failure is rankedhigh in the list of causes of deaths of cats.

The clinical symptoms of nephropathy, in particular chronic renalfailure, are such general or constitutional symptoms as polyposia andpolyuria, anorexia, weight loss, vomiting, diarrhea, dehydration,anemia, and depression. Diagnosis is carried out by or according to aninterview with the owner, urinalysis, blood examination, renal biopsy,and imaging, among others. As general indicators of nephropathy, theremay be mentioned blood urea nitrogen (hereinafter, BUN), serum creatinin(hereinafter, CRE), and proteinuria. However, accelerative increases inboth BUN and CRE are observed only after a decrease in glomerularfiltration value (an index in renal function evaluation) to 25% of thenormal level or below, hence they cannot serve as markers for earlydiagnosis. In addition, the BUN is influenced by meals, and the CRE byphysical activity. As for the urinary protein, or proteinuria, it is oneof findings suggesting disorders of kidneys and is so important thatwhen it is found, nephropathy is suspected first of all. However, whilethe protein excretion in normal human urine is 3 mg or less/kg/day, theprotein excretion in feline urine is as high in concentration as17.43±9.03 mg/kg/day even in normal conditions. Thus, the equationproteinuria=nephropathy can hardly be formulated.

Under such circumstances, the advent of a marker for early diagnosis offeline nephropathy is now earnestly desired.

The conventional methods of diagnosing nephropathy mainly utilize suchcomplicated techniques as blood examination, radiography of kidneysusing a contrast medium, ultrasonic image analysis, and so forth. It isdemanded that a more easy and simple method be developed. Further, it isdesired that a method by which the owner can check the health status ofa cat in a simple and easy manner without visiting a veterinarian and atest kit therefor be developed. A convenient test reagent is alsorequired with which feline nephropathy can easily be found before thedisease becomes incurable, with the result that the life of a family catcan be prolonged.

DISCLOSURE OF INVENTION

The present inventors have so far made investigations on the theme offeline nephropathy resulting from advanced age and, thus, concerning amethod of diagnosing feline nephropathy by urinary protein analysisutilizing SDS-PAGE. In the course thereof, they discovered a novelprotein (named cauxin by them) having a molecular weight of 70 K andexcreted in feline urine in high concentrations in normal adult catsshowing no clinical abnormality, and examined the concentrations ofcauxin excreted in urine in cats with various diseases. As a result, itwas confirmed that cats already in the state of renal failure showmarkedly decreased urinary concentrations of cauxin. Thus, theyproceeded with the analysis of cauxin as a novel marker for diagnosingnephropathy. Further, they determined the nucleotide base sequence ofthe cauxin gene and the amino acid sequence of cauxin and thus revealedthat the nucleotide base sequence determined has 47% homology with thecarboxylesterase family, that cauxin has carboxylesterase activity, andthat, unlike other carboxylesterases, it is expressed in vivospecifically in the epithelial cells of distal kidney tubules. Thus,they were led to a recognition that cauxin is not a serumprotein-derived urinary protein but a novel carboxylesterase-likeprotein produced in distal kidney tubule epithelial cells and secretedinto tubular cavities from the apical side and excreted into urine.Further investigations based on the utility as a marker for diagnosingfeline nephropathy have now led to completion of the present invention.

The novel diagnostic marker according to the present invention, which isproduced specifically in renal tubular tissues and excreted into urine,creates the possibility of renal tissue disorders being found at anearly stage and the possibility of remaining normal tubular tissues indiseased kidneys being quantitatively grasped. Therefore, it isconsidered to be a very useful item from the clinical viewpoint.

The present invention provides:

[1] A protein characterized by its being cauxin, or a salt thereof;

[2] A protein or a salt thereof as defined above under [1] which ischaracterized by its serving as a marker for diagnosing felinenephropathy;

[3] A protein or a salt thereof as defined above under [1] or [2] whichis characterized by

-   (a) having at least the sequence Asp²⁶ to Pro⁵⁴² out of the amino    acid sequence shown under SEQ ID NO: 2 in the sequence listing, or-   (b) having the amino acid sequence shown under SEQ ID NO: 2 in the    sequence listing, or-   (c) having at least 50% homology with the sequence Asp²⁶ to Pro⁵⁴²    out of the amino acid sequence shown under SEQ ID NO: 2 in the    sequence listing or having at least 50% homology with the amino acid    sequence shown under SEQ ID NO: 2 in the sequence listing;

[4] A partial peptide of the protein defined above under any of [1] to[3];

[5] A nucleic acid characterized by having a nucleotide base sequencecoding for the protein defined above under any of [1] to [3] or thepartial peptide defined above under [4];

[6] A nucleic acid as defined above under [5] which is characterized by

-   (a) having the nucleotide base sequence shown under SEQ ID NO: 1 in    the sequence listing,-   (b) having the nucleotide base sequence shown under SEQ ID NO: 3 in    the sequence listing,-   (c) having at least the sequence from the 250th nucleotide base to    the 1803rd nucleotide base out of the nucleotide base sequence shown    under SEQ ID NO: 3 in the sequence listing,-   (d) being capable of hybridizing with a sequence having any of the    nucleotide base sequences defined above under (a) to (c) under    stringent conditions, or-   (e) being capable of hybridizing with a nucleotide base sequence    comprising ten or more consecutive nucleotide bases out of the    nucleotide base sequence shown under SEQ ID NO: 1 or SEQ ID NO: 3 in    the sequence listing;

[7] A vector characterized by containing the nucleic acid defined aboveunder [5] or [6];

[8] A transformant characterized by harboring the nucleic acid definedabove under [5] or [6] or the vector defined above under [7];

[9] A method of obtaining the protein defined above under any of [1] to[3] or the partial peptide defined above under [4] which comprisescultivating the transformant defined above under [8] and recovering theproduct from the culture;

[10] An antibody characterized by being capable of specifically bindingto the protein defined above under any of [1] to [3] or the partialpeptide defined above under [4];

[11] A method of diagnosing feline nephropathy which comprisesquantitatively assaying for urinary cauxin and detecting felinenephropathy with using as an index a decrease in the level of cauxin;

[12] A method of diagnosing feline nephropathy as defined above under[11] which comprises

-   (i) the step of subjecting a urinary protein-containing sample to    separation by electrophoresis, or-   (ii) the step of assaying for a biological urinary cauxin activity,    or-   (iii) the step of bringing a urinary protein-containing sample into    contact with an anti-cauxin antibody;-   [13] A diagnostic agent for feline nephropathy which comprises-   (i) a cauxin-staining agent, or-   (ii) a reagent for assaying for a biological urinary cauxin    activity, or-   (iii) an anti-cauxin antibody;-   [14] A cauxin-detecting kit which comprises-   (a) a sample application site, (b) a labeled antibody-containing    site, (c) an antigen detection site and (d) a reaction completion    judging site, as disposed in that order on a carrier or support    enabling substances to move in a wet condition, wherein-   (i) the labeled antibody-containing site contains a labeled    anti-cauxin antibody (labeled antibody) which is capable of    migrating on the carrier to the antigen detection site and then to    the reaction completion judging site in a wet condition,-   (ii) an immobilized anti-cauxin antibody (immobilized antibody) is    placed on the detection site, and-   (iii) a site where an antibody (second antibody) to the antibody    used as the labeled antibody is immobilized is formed on the    reaction completion judging site, and wherein    when a sample is applied to the sample application site, the sample    is allowed to migrate on the carrier, elute the labeled antibody and    pass through the immobilized antibody on the antigen detection site    and through the second antibody site on the reaction completion    judging site, for detecting cauxin in the sample;

[15] A cauxin detecting kit for the assay of cauxin in a sample which ischaracterized in that a sample is brought into contact with a substratefor carboxylesterase and the resulting signal is measured; and

[16] A cauxin detecting kit characterized in

(1) that a sample is brought into contact with an immobilizedanti-cauxin antibody and then with a labeled anti-cauxin antibody or

(2) that a sample is brought into contact with a labeled anti-cauxinantibody and then an immobilized anti-cauxin antibody, and cauxin isassayed utilizing the label as an indicator.

Other objects, features, advantages and viewpoints of the presentinvention will become apparent to those skilled in the art in view ofthe description which follows. It is to be understood, however, that thedescription in the present specification, including the subsequentdescription and the description of specific examples, is given forillustrating preferred embodiments of the present invention and only forillustrative purposes alone. It will be quite obvious to those skilledin the art that various changes and/or improvements (or modifications)can be made within the spirit and scope of the present invention asdisclosed herein based on the subsequent description and the knowledgeobtainable from other portions of the present specification. All thepatent documents and references cited herein have been cited for thepurpose of illustration and the contents therein shall be construed asconstituting a part of and being contained in the present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the SDS-polyacrylamide electrophoresisprofiles of feline urinary proteins.

FIG. 2 is a photograph showing the SDS-polyacrylamide gelelectrophoresis profiles of proteins in urine samples derived fromanimals belonging to the family Felidae.

FIG. 3 shows the primary structure of cauxin, in comparison withcarboxylesterase.

FIG. 4 shows the C-terminal amino acid sequence alignment of cauxin asshown in comparison with carboxylesterases obtained from rat kidney, ratliver, swine liver and human liver.

FIG. 5 is a photography showing the patterns of western blotting,following electrophoresis, of protein samples extracted from the catserum, liver, kidney and pancreas.

FIG. 6 is a photograph showing the patterns of northern blotting,following electrophoresis, of RNA samples extracted from various catorgans using the cauxin cDNA.

FIG. 7 is a schematic representation of the kidney of an adult cat.

FIG. 8A is a tissue photograph illustrating the in situ hybridization(×50) patterns of the slanting line area shown in FIG. 7, and FIG. 8B isa tissue photograph illustrating the immunohistochemical staining (×50)patterns of the same area.

FIG. 9C is a tissue photograph illustrating the in situ hybridization(×100) patterns of a corticomedullar transitional zone of an adult catkidney, FIG. 9D is an enlargement (in situ hybridization (×400)) of thesite shown in FIG. 9C, and FIG. 9E is a tissue photograph illustratingthe in situ hybridization (×200) patterns of a corticomedullartransitional zone of an adult cat kidney.

FIG. 10 is a tissue photograph illustrating the immunohistochemicalstaining (×100) patterns of a corticomedullar transitional zone of anadult cat kidney.

FIG. 11 is an enlarged tissue photograph (immunohistochemical staining(×200)) of the site shown in FIG. 10.

FIG. 12 is a tissue photograph illustrating the results ofimmunohistochemical staining (×400) using an FITC-labeled antibody.

FIG. 13 is a tissue photograph illustrating the immune electronmicroscopy (×60,000) images of the endoplasmic reticulum in tubularepithelial cells.

FIG. 14 is a photograph showing the SDS-polyacrylamide electrophoresisprofiles of normal cat-derived and nephropathic cat-derived urinaryprotein samples.

FIG. 15 is a tissue photograph showing the staining patterns of a tissuederived from a cat with interstitial nephritis.

FIG. 16 is a tissue photograph showing the immunostaining patterns forcounting cauxin-producing cells in a corticomedullar transitional zoneof a normal (healthy) cat.

FIG. 17 is a tissue photograph showing the immunostaining patterns forcounting cauxin-producing cells in a corticomedullar transitional zoneof a cat with nephropathy.

BEST MODES FOR CARRYING OUT THE INVENTION

Cauxin occurs in high concentrations in urine under normal conditionswhile the urinary level of cauxin decreases with the decrease in renalfunctions. By taking into consideration the extent of decrease in itslevel, it becomes possible to estimate the amount of tissues performingnormal renal functions. Thus, it is desirable that the assay bequantitative, not qualitative. The sample to be used for cauxin assay isurine, and the cauxin concentration in urine is influenced by the amountof urine. Therefore, for avoiding the influence on the amount of urine,the ratio of cauxin to urinary creatinine or urinary protein, forinstance, is preferably determined.

In the practice of the present invention, the cauxin assaying kit, forinstance, is preferably so constituted that it can be used in the mannerof (A) screening, (B) diagnosis, or (C) routine control. The assayingmode may be either a simplified one or an exact one. In the simplifiedmode, the cat keepers, for instance, can be regarded as users, and thekit may be of the stick type enabling rough estimation of the cauxinlevel according to the extent of coloration, of the sheet type to belaid on the toilet for a cat, or of the small bead type to be used inadmixture with the sand for use in the toilet for a cat. In the exactmode, it is allowed that the user is restricted to a veterinarian, forinstance, and the kit may be of the liquid type for use in assaying onan automated analyzer.

The assay method may be either the one measuring the enzyme activity orthe one using the antigen-antibody interaction. In particular,techniques involving an immunological method are preferred.

The present invention provides cauxin polypeptide or a salt thereof, apeptide, inclusive of salts thereof, having at least 50% homology withthe amino acid sequence of the cauxin polypeptide and havingcarboxylesterase activity or comparable antigenicity, a partial peptide,inclusive of salts thereof, characteristic of the polypeptide, genescoding therefor, for example DNAs and RNAs, vectors or plasmidscontaining such a gene in a manner such that the gene can be manipulatedby the gene recombination technology, host cells transformed with such avector or the like, further transgenic animals, such as transgenic mice,in which such a gene is expressed, knockout animals, such as knockoutmice, in which such gene has been specifically inactivated, a method ofproducing the relevant polypeptide or a salt thereof by cultivating suchtransformant cells, antibodies, in particular monoclonal antibodies,obtained by using the thus-obtained polypeptide or a salt thereof, or acharacteristic partial peptide of that polypeptide or a salt thereof,hybridoma cells producing such an antibody, as well asassaying/diagnosing means and reagents which use such a gene isolated,for example a DNA or RNA, as a probe or use such an antibody.Furthermore, there are provided the uses of those active ingredientsdisclosed and described herein, for example pharmaceutical preparationsor reagents containing such active ingredients, and methods for thetreatment and/or prophylaxis of diseases, disorders or abnormalconditions using such active ingredients and, further, relevantscreening methods and so forth.

The term “cauxin” as used herein refers to a novel peptide found for thefirst time as a 70 K protein in the urine of adult cats and disclosed inthe present specification. Cauxin is a peptide composed of 542 aminoacid residues and is characterized in that it lacks the region ofendoplasmic reticulum retention signal (HXEL), which carboxylesteraseshave, on the C-terminal side. In view of its similarity tocarboxylesterases, it is estimated to have carboxylesterase activity andfurther estimated to have some or other activity in the metabolism oflipids in cats. Thus, cauxin includes, within the meaning thereof,species having carboxylesterase activity.

The term “polypeptide” as used herein may refer to any of thepolypeptides described later herein. The fundamental structures ofpolypeptides are well known in the art and are described in a very largenumber of reference books and other publications in the relevant fieldof art. In view of such situation, the term “polypeptide” as used hereinrefers to any peptide or any protein comprising two or more amino acidresidues bonded together via peptide bonding or modified peptidebonding. The term “polypeptide” as used herein includes both theshort-chain ones also referred to as peptides, oligopeptides or peptideoligomers and the long-chain ones generally referred to as proteins andknown to occur in various shapes and forms.

The polypeptide may often contain an amino acid(s) other than the 20amino acids generally called natural amino acids (naturally occurringamino acids; or amino acids encoded by genetic codes). It will beunderstood that a large number of amino acid residues, including theterminal amino group, of the polypeptide can be altered (modified) notonly by a natural step such as posttranslational processing and/or otheralteration (or modification) but also by some or other chemicalmodification technology well known in the art. Various alterations(modifications) applicable to the polypeptide are known and described indetail in basic reference books, detailed study reports and a largenumber of scientific documents and thus well known to those skilled inthe art. As several, in particular conventional,alterations/modifications, there may be mentioned glycosylation, lipidbinding, sulfation, γ-carboxylation with a glutamic acid residue,hydroxylation, and ADP-ribosylation, among others, and the descriptionsin T. E. Creighton, Proteins—Structure and Molecular Properties, SecondEdition, W. H. Freeman and Company, New York, (1993); B. C. Johnson(Ed.), Posttranslational Covalent Modification of Proteins, AcademicPress, New York, (1983) (Wold, F., “Posttranslational ProteinModifications: Perspective and Prospects”, pp. 1–12); Seifter et al.,“Analysis for Protein Modifications and nonprotein cofactors”, Meth.Enzymol. 182: 626–646 (1990); Rattan et al., “Protein Synthesis:Posttranslational Modification and Aging”, Ann. N.Y. Acad. Sci., 663:pp. 48–62 (1992), for instance, can be referred to.

The “polypeptide” of the present invention includes, among others,cauxin and related polypeptides. The cauxin and related polypeptidesinclude all of those derived from animals of the family Felidae, thosehaving carboxylesterase activity or activity involved in lipidmetabolism, typically those observed in normal adult cats (family cats),showing decreases in urinary excretion in kittens or cats suffering fromnephropathy and forming a band at about 70 K in SDS-PAGE and, morespecifically, those having an amino acid sequence which has at least 50%homology with the amino acid sequence shown under SEQ ID NO: 2 in thesequence listing and being substantially equivalent thereto inbiological activities such as carboxylesterase activity and/orantigenicity.

The “cauxin” of the present invention may be one having carboxylesteraseactivity, or one having a novel amino acid sequence highly homologouswith the amino acid sequence shown under SEQ ID NO: 2 in the sequencelisting. As more preferred peptides of the present invention, there maybe mentioned those having 10 or more, preferably 20 or more, consecutiveamino acid residues selected from the amino acid sequence shown underSEQ ID NO: 2 in the sequence listing. The peptide of the presentinvention may have part or all of the amino acid sequence shown underSEQ ID NO: 2 in the sequence listing, and includes all of those havingsuch a sequence. As preferred examples, there may be mentioned thosehaving at least the sequence from Asp²⁶ to Pro⁵⁴², or a part thereof,out of the sequence shown under SEQ ID NO: 2 in the sequence listing.

In the present specification, “homology” refers to the quantity (number)of those corresponding amino acid residues or nucleotide bases betweentwo polypeptide sequence (or amino acid sequence) or polynucleotidesequence (or base sequence) chains which can be judged as the same intheir matching relationship, and thus means the degree of sequencesimilarity or relatedness between two polypeptide sequences or twopolynucleotide sequences. The homology can easily be calculated. Anumber of methods of determining the homology between two polynucleotidesequences or polypeptide sequences are known, and the term “homology”(also referred to as “similarity”) is well known in the art (e.g. Lesk,A. M. (Ed.), Computational Molecular Biology, Oxford University Press,New York, (1988); Smith, D. W. (Ed.), Biocomputing; Informatics andGenome Projects, Academic Press, New York, (1993); Grifin, A. M. &Grifin, H. G. (Ed.), Computer Analysis of Sequence Data: Part I, HumanPress, New Jersey, (1994); von Heinje, G., Sequence Analysis inMolecular Biology, Academic Press, New York, (1987); Gribskov, M. &Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, NewYork, (1991), etc.).

The general methods to be used in determining the homology between twosequences include, but are not limited to, those disclosed in Martin, J.Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego,(1994); Carillo, H. & Lipman, D., SIAM J. Applied Math., 48: 1073(1988), and so forth. Preferred as the method of homology estimation isthe one which has been designed to find out the maximum matchingrelation between two sequences to be tested. As such method, there maybe mentioned those formulated as computer programs. Preferred computerprograms for estimating the homology between two sequences include, butare not limited to, the GCG program package (Devereux, J. et al.,Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA(Atschul, S. F. et al., J. Molec. Biol., 215: 403 (1990)), etc. Any ofthose known in the relevant field of art can be used.

In accordance with the present invention, the gene coding for cauxin istypically one having a nucleotide base sequence coding for the peptideshown under SEQ ID NO: 2 in the sequence listing or a partialconsecutive amino acid sequence thereof, for example one having thenucleotide base sequence shown under SEQ ID NO: 1 in the sequencelisting, or may be one resulting from addition of an initiation codon,for example the codon coding for Met (and a termination codon), to suchnucleotide base sequence, one having a nucleotide base sequence codingfor a peptide having an amino acid sequence showing at least 80%homology with the protein encoded by the nucleotide base sequencementioned above and having carboxylesterase activity or a peptidesubstantially equivalent in biological activities, for exampleantigenicity, thereto, or one having a nucleotide base sequenceequivalent to such nucleotide base sequence. The cauxin-encoding genemay be in the form of single-stranded DNA, double-stranded DNA, RNA,DNA:RNA hybrid, synthetic DNA or a like nucleic acid, and may be a cDNAderived from feline genomic DNA, a feline genomic DNA library, ortissues or cells of an animal of the family Felidae, or a synthetic DNA.The nucleotide base sequence of the cauxin-encoding gene may be modified(e.g. by addition, deletion or substitution), and the gene in questionmay include such modifications. As described later herein, the nucleicacid of the present invention may also be one coding for the peptide ofthe present invention or a part thereof, and preferably is a DNA. As theabove-mentioned “equivalent nucleotide base sequence”, there may bementioned those capable of hybridizing with a nucleotide base sequencecomprising 10 or more, preferably 20 or more consecutive nucleotidebases selected from the nucleotide base sequence shown under SEQ ID NO:1 or SEQ ID NO: 3 in the sequence listing under stringent conditions andcoding for an amino acid sequence substantially equivalent to cauxin.

In accordance with the present invention, gene recombinationtechnologies can be used to isolate and sequence the desired nucleicacid, construct recombinants thereof, and produce the desired peptide.The gene recombination technologies (inclusive of recombinant DNAtechnologies) can be carried out by the methods described, for example,in J. Sambrook, E. F. Fritsch & T. Maniatis, “Molecular Cloning: ALaboratory Manual (2nd edition)”, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); D. M. Glover et al. ed., “DNA Cloning”,2nd ed., Vols. 1 to 4, (The Practical Approach Series), IRL Press,Oxford University Press (1995); Japanese Biochemical Society (ed.),“Zoku Seikagaku Jikken Koza (Experiments in Biochemistry, Second Series)1, Idenshi Kenkyuho (Methods of Gene Studies) II”, Tokyo Kagaku Dozin(1986); Japanese Biochemical Society (ed.), “Shin Seikagaku Jikken Koza(Experiments in Biochemistry, Updated) 2, Kakusan (Nucleic Acids) III(Recombinant DNA Technology)”, Tokyo Kagaku Dozin (1992); R. Wu ed.,“Methods in Enzymology”, Vol, 68 (Recombinant DNA), Academic Press, NewYork (1980); R. Wu et al. ed., “Methods in Enzymology”, Vol. 100(Recombinant DNA, Part B) & Vol. 101 (Recombinant DNA, Part C), AcademicPress, New York (1983); R. Wu et al. ed., “Methods in Enzymology”, Vol.153 (Recombinant DNA, Part D), Vol. 154 (Recombinant DNA, Part E) & Vol.155 (Recombinant DNA, Part F), Academic Press, New York (1987); J. H.Miller ed., “Methods in Enzymology”, Vol. 204, Academic Press, New York(1991); R. Wu et al. ed., “Methods in Enzymology”, Vol. 218, AcademicPress, New York (1993); S. Weissman (ed.), “Methods in Enzymology”, Vol.303, Academic Press, New York (1999); and J. C. Glorioso et al. (ed.),“Methods in Enzymology”, Vol. 306, Academic Press, New York (1999), ormethods substantially identical thereto, or modifications thereof (therelevant descriptions in these references are incorporated herein byreference) [hereinafter all of these are collectively referred to as“gene recombination technology”].

The term “polymerase chain reaction” or “PCR” as used herein generallyrefers to such a method as described in U.S. Pat. No. 4,683,195 and, forexample, refers to a method for enzymatically amplifying a desiredoligonucleotide sequence in vitro. Generally, PCR comprises repetitionsof a cycle for nucleotide sequence synthesis by primer extension usingtwo oligonucleotide primers capable of preferentially hybridizing with atemplate nucleic acid. Typically, the primers to be used in PCR may beprimers complementary to the nucleotide sequence to be amplified in thetemplate and, for example, those complementary at both ends of thenucleotide sequence to be amplified or adjacent to the nucleotidesequence to be amplified are preferably used. The 5′ end side primer ispreferably selected so that it contains at least the initiation codon oramplification inclusive of the initiation codon can be carried out,while the 3′ end side primer is preferably selected so that it containsat least the stop codon or amplification inclusive of the stop codon canbe carried out. Preferred as the primers are oligonucleotides comprising18 to 25 nucleotide bases. The primers can be prepared by the methodswell known in the art. Typically, they can be chemically synthesized bythose known methods described in Angew. Chem. Int. Ed. Engl., Vol. 28,pp. 716–734 (1989), for example the phosphotriester method,phosphodiester method, phosphite method, phosphoamidite method, andphosphonate method. It is generally known that such synthesis can beconveniently carried out on a modified solid supporting member using,for example, an automated synthesizer, such as a model 381A DNAsynthesizer (Applied Biosystems). The oligonucleotides may contain oneor more modified nucleotide bases, for example inosine or a likenucleotide base nonconventional in the nature, or a tritylatednucleotide base.

The PCR can be carried out by the methods known in the art, methodssubstantially identical thereto, or modifications thereof, for examplethe method described in R. Saiki, et al., Science, 230: 1350, 1985; R.Saiki, et al., Science, 239: 487, 1988; H. A. Erlich ed., PCRTechnology, Stockton Press, 1989; D. M. Glover et al. ed., “DNACloning”, 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press,Oxford University Press (1995); M. A. Innis et al. ed., “PCR Protocols:a guide to methods and applications”, Academic Press, New York (1990);M. J. McPherson, P. Quirke and G. R. Taylor (Ed.), PCR: a practicalapproach, IRL Press, Oxford (1991); M. A. Frohman et al., Proc. Natl.Acad. Sci. USA, 85, 8998–9002 (1988), etc., or modifications or improvedversions thereof. The PCR method can also be carried out usingcommercially available kits suited therefor according to the protocolsdisclosed by kit manufacturers or kit distributors.

The PCR product obtained is generally subjected to 1 to 2% agarose gelelectrophoresis, specific bands are excised from the gel, and DNA isextracted using a commercial extraction kit such as a gene clean kit(Bio 101), for instance. The DNA extracted is cleaved with anappropriate restriction enzyme(s) and, after purification treatment,and, if necessary, after further phosphorylation at the 5′ end using T4polynucleotide kinase or the like, the fragment obtained is ligated toan appropriate vector such as pUC18 or a like pUC vector for use intransformation of appropriate competent cells. The PCR product clones isanalyzed for its nucleotide base sequence. For cloning the PCR product,such commercial plasmid vectors as p-Direct (Clontech), pCR-Script™SK(+) (Stratagene), pGEM-T (Promega) and pAmp™ (Gibco-BRL), forinstance, can be used. For transforming host cells, those methods knownin the relevant field of art or methods substantially equivalent theretocan be used, for example using a phage vector or by the calcium method,rubidium/calcium method, calcium/manganese method, TFB high efficiencymethod, FSB frozen competent cell method, rapid colony method, orelectroporation (D. Hanahan, J. Mol. Biol., 166: 557, 1983, etc.). Forisolating the desired DNA, polymerase chain reaction coupled reversetranscription (RT-PCT) or RACE (rapid amplification of cDNA ends) can beapplied. The RACE can be carried out according to the method describedin M. A. Innis et al. ed., “PCR Protocols” (M. A. Frohman, “a guide tomethods and applications”), pp. 28–38, Academic Press, New York (1990),for instance.

For nucleic acid identification, for instance, such a hybridizationtechnique as mentioned below is utilized.

The DNA obtained can be cloned, if necessary, and, for example, aplasmid, λ phage, cosmid, P1 phage, F factor, YAC or the like can beused for that purpose. Preferred are λ phage-derived vectors and, thus,Charon 4A, Charon 21A, λgt10, λgt11, λDASHII, λFUXII, λEMBL3, λZAPII™(Stratagene) and the like can be utilized. Further, the DNA obtained maybe inserted into an appropriate vector, for example the plasmid pEX,pMAMneo or pKG5, for expression thereof in appropriate host cells, suchas Escherichia coli, yeast cells, CHO cells, or COS cells, as explainedlater herein in detail. Further, the DNA fragment, either as it is or asa DNA fragment resulting from addition of an appropriate regulatorysequence, can be inserted into an appropriate vector and then introducedinto an animal to produce a transgenic animal in which the desired gene,for example the propeptide-deficient pro-MMP-7 or C terminal-deficientCD151, is expressed. As such animal, there may be mentioned mammals suchas mice, rats, rabbits, guinea pigs and bovine species. Preferably, thegene fragment can be introduced into fertilized animal (e.g. mouse) eggsto produce transgenic animals. The foreign gene can be introduced intomammalian or like animal cells by the methods known in the art or methodsubstantially equivalent thereto, for example by the calcium phosphatemethod (e.g. F. L. Graham et al., Virology, 52: 456, 1973), DEAE-dextranmethod (e.g. D. Warden et al., J. Gen. Virol., 3: 371, 1968),electroporation (e.g. E. Neumann et al., EMBO J., 1:841, 1982),microinjection, ribosome method, viral infection, phage particle method,etc. The gene product produced by animal cells thus transfected with theintended gene can also be analyzed.

The plasmid into which the intended gene (e.g. DNA obtained in accordingto the present invention) is to be inserted may be any of those plasmidswhich allow the DNA to be expressed in host cells commonly used ingenetic engineering (e.g. prokaryotic host cells such as Escherichiacoli or Bacillus subtilis, eukaryotic host cells such as yeasts, CHOcells or COS cells, or insect cells such as Sf21). Such a sequence maycontain, for example, a codon(s) adequately modified for the expressionin the host cells selected, a restriction enzyme site, a regulatorysequence and a promoter sequence for facilitating the expression of thegene in question, a linker(s) or adapter(s) serving in ligation of thegene in question, and, further, a sequence (including a sequence codingfor a hybrid protein or a fused protein) useful in regulating orcontrolling an antibiotic resistance or metabolism or in selection. Anappropriate promoter may preferably be used, for example the tryptophanpromoter (trp), lactose promoter (lac), tryptophan lactose promoter(tac), lipoprotein promoter (lpp), λ phage P_(L) promoter or the like inplasmids for which Escherichia coli cells serve as hosts, the SV40 latepromoter, MMTV LTR promoter, RSV LTR promoter, CMV promoter, SR αpromoter or the like in plasmids for which animal cells serve as hosts,or the GAL1 or GAL10 promoter or the like in plasmids for which yeastcells serve as hosts.

As plasmids for which Escherichia coli serves as a host, there may bementioned, for example, pBR322, pUC18, pUC19, pUC118, pUC119, pSP64,pSP65, pTZ-18R/-18U, pTZ-19R/-19U, pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z,pGEM-5Zf(−), pBluescript KS™ (Stratagene), etc. As plasmid vectorssuited for expression in Escherichia coli, there may also be mentionedpAS, pKK223 (Pharmacia), pMC1403, pMC931, pKC30, pRSET-B (Invitrogen),etc. As plasmids for which animal cells serve as hosts, there may bementioned the SV40 vector, polyoma virus vectors, vaccinia virusvectors, retrovirus vectors and the like, for example pcD, pcD-SRα,CDM8, pCEV4, pME18S, pBC12BI, pSG5 (Stratagene), etc. As plasmids forwhich yeasts serve as hosts, there may be mentioned YIp type vectors,YEp type vectors, YRp type vectors, YCp type vectors and the like, forexample pGPD-2. As for the host cells, when the host is Escherichiacoli, there may be mentioned Escherichia coli K12 strain-derived strainssuch as NM533, XL1-Vlue, C600, DH1, DH5, DH11S, DH12S, DH5α, DH10B,HB101, MC1061, JM109 and STBL2, and BL21(DE3)pLysS and the like as B834stain-derived ones. When the host cells are animal cells, there may bementioned, for example, African green monkey fibroblast-derived COS-7cells, COS-1 cells, CV-1 cells, mouse fibroblast-derived COP cells, MOPcells, WOP cells, Chinese hamster cell-derived CHO cells, CHO DHFR⁻cells, human HeLa cells, murine cell-derived C127 cells, murinecell-derived NIH 3T3 cells, etc. As for the insect cells, mention may bemade of the use of silkworm larvae or cultured silkworm cells, forexample BM-N cells, together with Bombyx mori nuclear polyhedrosis virusor a derivative thereof as the vector. It is also possible to use plantcells as host cells, which, together with adequate vectors, are widelyknown in the relevant field of art. In the genetic engineering techniqueaccording to the present invention, use can be made of those restrictionenzymes, reverse transciptases, DNA modifying or decomposing enzymes formodifying or converting DNA fragments to structures suited for cloning,DNA polymerases, terminal nucleotidyl transferases, DNAs and ligaseswhich are known or in wide use in the relevant field.

In accordance with the present invention, a cell line having high andstable expression capacity can be obtained by subjecting a transformantobtained by transformation with an expression vector containing aprotein-encoding nucleic acid to repeated cloning using an appropriateselective marker as necessary. For example, when the dhfr gene isutilized as the selective marker in transformant cells derived fromanimal cells used as host cells, a cell line capable of amplifying a DNAcoding for the protein according to the invention and thus causing ahigher level of expression can be obtained by carrying out cultivationwhile the MTX concentration is gradually increased and selecting aresistant strain. When cultured under conditions enabling the expressionof a nucleic acid coding for the protein of the present invention, thetransformant of the present invention can produce and accumulate thedesired product therein. For cultivating a transformant derived from aprokaryotic cell host such as Escherichia coli or Bacillus subtilis orfrom a yeast, for instance, a liquid medium can suitably be used. Themedium is caused to contain a carbon source(s), a nitrogen source(s),inorganic substances and so forth which are necessary for the growth ofthe transformant. The carbon source includes, among others, glucose,dextrin, soluble starch, and sucrose, the nitrogen source includes,among others, ammonium salts, nitrate salts, corn steep liquor, peptone,casein, meat extract, malt extract, soybean cake, potato extract andlike inorganic or organic substances, the inorganic substances include,among others, calcium chloride, sodium dihydrogen phosphate, magnesiumchloride, and calcium carbonate. Yeasts, vitamins, casamino acids,growth promoters and the like may also be added. If necessary, for moreefficient promoter functioning, such an agent as 3 β-indolylacrylicacid, for instance, may be added. The medium preferably has a pH ofabout 5 to 8.

In the case of Escherichia coli, for instance, the cultivation isgenerally carried out at about 15 to about 45° C. for about 3 to about75 hours. If necessary, aeration and/or stirring may be carried outadditionally. For cultivating a transformant derived from animal cellsas host cells, the medium to be used is, for example, about 5 to about20% fetal bovine serum-containing MEM medium, PRMI 1640 medium, or DMEMmedium. The pH is preferably about 6 to about 8. The cultivation isgenerally carried out at about 30° C. to about 40° C. for about 15 toabout 72 hours, if necessary with aeration and/or stirring. Thetransformant expressing the desired gene product may be utilized as itis or in the form of a cell homogenate, or the desired gene product canbe isolated for use thereof. In extraction from the cells cultured inthe above manner, any adequate method be used. For example, aftercultivation, the microbial or other cells are collected by aconventional method, suspended in an appropriate buffer solution, anddisrupted by sonication, with lysozyme and/or by freezing and thawing,for instance, and a crude extract is recovered by centrifugation orfiltration. To the buffer solution, there may be added a proteindenaturing agent such as urea or guanidine hydrochloride, and/or asurfactant such as Triton X-100 (trademark) or Tween 20 (trademark). Incases where the desired product is secreted into the culture fluid, theculture fluid after completion of cultivation is separated into themicrobial or other cells and the supernatant by a per se known method,and the supernatant is collected. The desired product contained in thethus-obtained culture supernatant or extract can be purified by anappropriate combination of per se known methods of separation andpurification, for example salting out with ammonium sulfate, forinstance, gel filtration using Sephadex, ion exchange chromatographyusing a carrier having diethylaminoethyl or carboxymethyl groups, forinstance, hydrophobic chromatography using a carrier having suchhydrophobic groups as butyl, octyl, or phenyl groups, dye gelchromatography, electrophoresis, dialysis, ultrafiltration, affinitychromatography, and high-performance liquid chromatography. Preferably,the product can be purified and isolated by such treatment aspolyacrylamide gel electrophoresis, or affinity chromatography utilizingan immobilized ligand or the like. Thus, gelatin-agarose affinitychromatography and heparin-agarose chromatography may be mentioned amongothers.

Further, mutant proteins corresponding to the protein in question asresulting from substitution, deletion, insertion, translocation and/oraddition of one or a plurality of amino acid residues can be producedbased on the nucleotide base sequence of the gene according to thepresent invention by using a method(s) generally used in geneticengineering. As the methods of such mutation, conversion ormodification, there may be mentioned the methods described, or example,in Japanese Biochemical Society (ed.), “Zoku Seikagaku Jikken Koza(Experiments in Biochemistry, Second Series) 1, Idenshi Kenkyuho(Methods of Gene Studies) II”, p. 105 (Susumu Hirose), Tokyo KagakuDozin (1986); Japanese Biochemical Society (ed.), “Shin Seikagaku JikkenKoza (Experiments in Biochemistry, Updated) 2, Kakusan (Nucleic Acids)III (Recombinant DNA Technology)”, p. 233 (Susumu Hirose), Tokyo KagakuDozin (1992); R. Wu, L. Grossman, ed., “Methods in Enzymology”, Vol.154, p. 350 & p. 367, Academic Press, New York (1987); R. Wu, L.Grossman, ed., “Methods in Enzymology”, Vol. 100, p. 457 & p. 468,Academic Press, New York (1983); J. A. Wells et al., Gene, 34: 315,1985; T. Grundstroem et al., Nucleic Acids Res., 13: 3305, 1985; J.Taylor et al., Nucleic Acids Res., 13: 8765, 1985; R. Wu, ed., “Methodsin Enzymology”, Vol. 155, p. 568, Academic Press, New York (1987); andA. R. Oliphant et al., Gene, 44: 177, 1986. For example, there may bementioned such methods as site-directed mutagenesis (site-specificmutagenesis) using a synthetic oligonucleotide (Zoller et al., Nucl.Acids Res., 10: 6487, 1987; Carter et al., Nucl. Acids Res., 13: 4331,1986), cassette mutagenesis (Wells et al., Gene, 34: 315, 1985),restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc.London Ser. A, 317: 415, 1986), alanine scanning (Cunningham & Wells,Science, 244: 1081–1086, 1989), PCR mutagenesis, Kunkel method, dNTP[αS]method (Eckstein), and region-directed mutagenesis using sulfurous acidor nitrous acid.

In producing by the gene recombination technology, it is also possibleto cause expression of the desired protein in the form of a fusedprotein. The fused protein thus obtained can be purified by affinitychromatography, for instance, utilizing the fused portion thereof. Assuch fused protein, there may be mentioned one fused to histidine tag,or to β-galactosidase (β-gal), maltose-binding protein (MBP),glutathione-S-transferase (GST), thioredoxin (TRX) or Cre recombinaseamino acid sequence. Similarly, it is also possible to add aheterogeneous epitope tag to the polypeptide for enabling purificationby immunoaffinity chromatography using an antibody specifically bindingto the epitope. As the epitope tag to be used in a more suitedembodiment, there may be mentioned, for example, AU5, c-Myc, CruzTag 09,CruzTag 22, CruzTag 41, Glu-Glu, HA, HA.11, KT3, FLAG (registeredtrademark; Sigma-Aldrich), Omni-probe, S-probe, T7, Lex A, V5, VP16,GAL4, and VSV-G (e.g. Field et al., Molecular and Cellular Biology, 8:pp. 2159–2165 (1988); Evan et al., Molecular and Cellular Biology, 5:pp. 3610–3616 (1985); Paborsky et al., Protein Engineering, 3 (6): pp.547–553 (1990); Hopp et al., BioTechnlogy, 6: pp. 1204–1210 (1988);Martin et al., Science, 255: pp. 192–194 (1992); Skinner et al., J.Biol. Chem., 266: pp. 15163–15166 (1991); Lutz-Freyermuth et al., Proc.Natl. Acad. Sci. USA, 87: pp. 6393–6397 (1990)).

The two-hybrid method using a yeast can also be utilized. Further, thefused protein may be one resulting from addition of a marker to renderthe protein detectable. In a more preferred embodiment, the detectablemarker may be Biotin Avi Tag of the biotin/streptavidin system, or afluorescent substance, for instance. The fluorescent substance includes,among others, an Aequorea victorea- or like fluorescentjellyfish-derived green fluorescent protein (GFP), variants thereof (GFPvariants), for example EGFP (enhanced-humanized GFP), rsGFP (red-shiftGFP), yellow fluorescent protein (YFP), green fluorescent protein (GFP),cyan fluorescent protein (CFP), blue fluorescent protein (BFP), andRenilla reniformis-derived GFP (Atsushi Miyawaki (ed.), Jikken Igaku(Experimental Medicine), an extra number, Posutogenomu Jidai no JikkenKoza (Experiments in Postgenomic Era) 3-GFP and Bioimaging, Yodosha(2000)). Detection can also be made using an antibody (including amonoclonal antibody and a fragment thereof) specifically recognizing theabove-mentioned fused tag. The protein obtained (which may include apeptide or polypeptide) can be immobilized by biding the same to anappropriate carrier or solid phase by a known technique such as anenzyme immunoassay technique. The immobilized protein or immobilizedpeptide can conveniently be used in binding assays or screening ofsubstances.

The protein obtained (which may include a peptide or polypeptide) can beused for screening purposes with its biological activity as anindicator. For example, it is possible to react the protein with asubstrate of a known carboxylesterase, for example p-nitrophenylaceticacid, and assay cauxin by measuring the color developed by thedecomposition product, since cauxin is considered to havecarboxylesterase activity. Typically, for instance, a filter paper orthe like is impregnated with an aqueous solution of p-nitrophenylaceticacid and then immersed in a feline urine sample for detecting a redcolor developed by the product formed upon decomposition bycarboxylesterase activity.

It is evident that the cauxin gene codes for a novel polypeptide. Hence,recombinant plasmids constructed using the cauxin gene are all novelrecombinants, and transformants or transfectants obtained bytransformation or transfection with the plasmids are also novel.

In the present specification, the term “substantially equivalent” meansthat the activities of a protein, for example its enzyme activity,physiological activity and biological activity, are substantially thesame as those of another protein. The term also includes, within themeaning thereof, the case where a protein has an activity ofsubstantially the same nature as compared with another. As the activityof substantially the same nature, there may be mentioned, for example,carboxylesterase activity, activity serving as a marker for felinenephropathy, for example glomerulopathy or interstitial lesion, anddecomposing activity against synthetic substrates of carboxylesterases.The activities of substantially the same nature indicate that theactivities are each qualitatively of the same nature, for examplephysiologically, pharmacologically or biologically, as compared with thecounterpart. For example, it is preferred that the activities, such ascauxin activity and carboxylesterase activity, be equivalent (about 0.1to about 10 times). However, the levels of these activities, themolecular weight of the protein and other quantitative factors maydiffer.

For synthesizing the protein of the present invention and partialpeptides thereof, methods known in the field of peptide synthesis, forexample liquid phase, solid phase and like chemical methods ofsynthesis, can be used. According to such methods, a resin for proteinor peptide synthesis, for instance, is used, and adequately protectedamino acids are subjected to condensation one by one on the resinaccording to the desired amino acid sequence by any of various per seknown condensation methods. In the condensation reaction, various per seknown activating reagents are preferably used. Preferred as suchreagents are carbodiimides such as dicyclohexylcarbodiimide. When theproduct has a protective group(s), deprotection is carried out in anappropriate manner to give the desired product.

The protein of the present invention and partial peptides thereof, whenobtained in the free form, may be converted to salts by a per se knownmethod or a modification thereof and, when obtained in a salt form, maybe converted to the free form or to other salts by a per se known methodor a modification thereof. The salts of the protein of the presentinvention and partial peptides thereof preferably include, but are notlimited to, physiologically acceptable ones or pharmaceuticallyacceptable ones. Such salts may include salts with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid andphosphoric acid, and salts with organic acids such as acetic acid,formic acid, maleic acid, fumaric acid, succinic acid, citric acid,tartaric acid, malic acid, benzoic acid, methanesulfonic acid,p-toluenesulfonic acid and benzene sulfonic acid. The salts may furtherinclude ammonium salts, salts with organic nucleotide bases such asethylamine, dimethylamine, trimethylamine and hydroxylamine, and so on.

In an embodiment, the feline nephropathy diagnosing agent of the presentinvention contains an anti-cauxin antibody as an active component. Theanti-cauxin antibody can be obtained as a polyclonal or monoclonalantibody by per se known means.

In the present specification, the term “antibody” may be used in a broadsense. Thus, it includes individual monoclonal antibodies to the desiredcauxin polypeptide and related peptide fragments, and antibodycompositions having specificity for various epitopes. It includesunivalent antibodies and polyvalent antibodies as well as monoclonalantibodies and polyclonal antibodies and, further, the native (intact)molecules, and fragments and derivatives thereof. Thus, it includes suchfragments as F(ab′)₂, Fab′ and Fab and, further, chimera antibodies orhybrid antibodies having at least two antigen or epitope binding sites,or bispecific recombinant antibodies such as quadromes and triomes,interspecific hybrid antibodies, anti-idiotype antibodies and, further,chemically modified or processed ones which can be considered to bederivatives of these, antibodies obtained by applying a per se knowncell fusion or hybridoma technology or antibody engineering technologyor utilizing a synthetic or semisynthetic technology, antibodiesprepared by applying a prior art technology known from the antibodyformation viewpoint or using a recombinant DNA technology, andantibodies having neutralizing activity or binding activity against thetarget antigen substance or target epitope as described and definedherein.

A particularly preferred antibody according to the present invention canspecifically recognize the native type cauxin polypeptide. For obtainingthe anti-cauxin antibody as a polyclonal antibody, a mammal or bird, forinstance, is immunized with the immunogen cauxin or a fragment thereof,or a partial peptide of the cauxin sequence, and antiserum is collectedfrom the mammal or bird. The polyclonal antibody contained in thisantiserum can then be used. The mammal to be immunized with thissensitizing antigen cauxin is not particularly restricted but,generally, rodents such as mice, rats, and hamsters and, further,rabbits, sheep, goats, cattle, horses, pigs, dogs, monkeys and otherprimates, and birds such as chickens are used. In some cases, it ispreferred that the mammal be selected considering the compatibility withthe parent cells to be used in cell fusion.

The immunization of animals with the sensitizing antigen is carried outby a per se known method. For example, a method in general use comprisesintraperitoneally or subcutaneously injecting the sensitizing antigeninto the mammals or the like. In immunization with the sensitizingantigen, an appropriate carrier may be used. After a predeterminedperiod of feeding of the immunized animals, the polyclonalantibody-containing antiserum can be prepared from the blood collectedfrom each of the animals. After confirming that it specificallyrecognizes cauxin, the antiserum obtained is submitted to use as adiagnostic agent for feline nephropathy.

Cauxin, which is to be used as the sensitizing antigen for antibodyproduction, can be obtained by causing expression of the cauxingene/amino acid sequence disclosed herein.

In the practice of the present invention, the anti-cauxin antibody mayalso be one obtained as a mammal-derived monoclonal antibody.

The monoclonal antibody produced against the antigenic substance can beproduced by any of the methods capable of causing the production ofantibody molecules in a series of cell lines under cultivation. Themodifier “monoclonal” indicates the characteristic of an antibody thatit belongs to a substantially homogeneous antibody population. It is notto be construed that the antibody should be produced by a certainspecific method. Individual monoclonal antibodies each includes apopulation of the same antibodies except that a slight amount of amutant(s) possible formed spontaneously may be present therein.Monoclonal antibodies each has high specificity and is directed to onesingle antigenic site. As compared with ordinary (polyclonal) antibodypreparations typically containing various antibody species directed todifferent antigenic determinants (epitopes), each monoclonal antibody isdirected to one single antigenic determinant on the antigen. In additionto their specificity, monoclonal antibodies are synthesized by hybridomaculture and are superior in that they are not or only a littlecontaminated with other immunoglobulins. The monoclonal antibodiesinclude hybrid antibodies and recombinant antibodies. So long as theyshow the desired biological activities, a constant region domain may besubstituted for a variable region domain thereof, or a heavy chain maybe substituted for a light chain thereof, a chain derived from a certainspecies may be replaced with a chain derived from another species, orthey may be fused with a heterogeneous protein, irrespective of theirorigin or immunoglobulin class or subclass (e.g. U.S. Pat. No.4,816,567; Monoclonal Antibody Production Techniques and Applications,pp. 79–97, Marcel Dekker, Inc., New York, 1987).

As preferred examples of the monoclonal antibody production, there maybe mentioned the hybridoma method (G. Kohler and C. Milstein, Nature,256, pp. 495–497 (1975)); human B cell hybridoma method (Kozbor et al.,Immunology Today, 4, pp. 72–79 (1983); Kozbor, J. Immunol., 133, pp.3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51–63, Marcel Dekker, Inc., New York (1987));trioma method; EBV-hybridoma method (Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77–96 (1985)) (methods ofproducing monoclonal antibodies); U.S. Pat. No. 4,946,778 (technology ofproducing single-chain antibodies) and, further, the followingreferences may be cited: S. Biocca et al., EMBO J, 9, pp. 101–108(1990); R. E. Bird et al., Science, 242, pp. 423–426 (1988); M. A. Bosset al., Nucl. Acids Res., 12, pp. 3791–3806 (1984); J. Bukovsky et al.,Hybridoma, 6, pp. 219–228 (1987); M. DAINO et al., Anal. Biochem., 166,pp. 223–229 (1987); J. S. Huston et al., Proc. Natl. Acad. Sci. USA, 85,pp. 5879–5883 (1988); P. T. Jones et al., Nature, 321, pp. 522–525(1986); J. J. Langone et al. (ed.), “Methods in Enzymology”, Vol. 121(Immunological Techniques, Part I: Hybridoma Technology and MonoclonalAntibodies), Academic Press, New York (1986); S. Morrison et al., Proc.Natl. Acad. Sci. USA, 81, pp. 6851–6855 (1984); V. T. Oi et al.,BioTechniques, 4, pp. 214–221 (1986); L. Riechmann et al., Nature, 332,pp. 323–327 (1988); A. Tramontano et al., Proc. Natl. Acad. Sci. USA,83, pp. 6736–6740 (1986); C. Wood et al., Nature, 314, pp. 446–449(1985); Nature 314, pp. 452–454 (1985); and references cited therein(the descriptions therein are incorporated herein by reference).

The monoclonal antibody according to the present invention includes“chimera” antibodies (immunoglobulins), in particular, in which part ofthe heavy chain and/or light chain has a sequence identical orhomologous to the corresponding sequence of an antibody derived from aspecific species or belonging to a specific antibody class or subclasswhile the remaining portions are identical or homologous to thecorresponding sequences of an antibody derived from another species orbelonging to another antibody class or subclass, so long as they showthe desired biological activities (U.S. Pat. No. 4,816,567; Morrison etal., Proc. Natl. Acad. Sci. USA, 81, pp. 6851–6855 (1984)).

The monoclonal antibody according to the present invention includesthose produced by mammal-derived hybridomas, and those produced by hoststransformed with an expression vector containing the antibody geneutilizing the genetic engineering technology.

Monoclonal antibody-producing hybridomas capable of producinganti-cauxin antibodies can be prepared by utilizing the cell fusiontechnology using myeloma cells, as follows. Thus, they can be preparedby carrying out immunization with a conventional method using cauxin ora fragment thereof as a sensitizing antigen, fusing the immunized cellsobtained with per se known parent cells by a conventional cell fusionmethod, and screening for monoclonal antibody-producing cells by aconventional screening method.

As for the method of preparing cauxin or a fragment thereof and themethod of immunizing mammals, among others, the techniques mentionedabove for the preparation of polyclonal antibody-containing antisera canbe employed. In this case, when mammals, in particular, are immunized,immunized cells are collected from the mammals after confirming anincrease in the serum level of the desired antibody, and they aresubjected to cell fusion. Spleen cells, in particular, are preferred asthe immunized cells.

Mammalian myeloma cells are used as counterpart parent cells to be fusedwith the above immunized cells. Various known cell lines can be used asthe myeloma cells. The cell fusion between the immunized cells andmyeloma cells and other steps can be carried out basically by knownmethods, for example by the method of Kohler and Milstein (Kohler, G.and Milstein, C., Methods Enzymol. (1981), 73, 3–46).

In the following, the production of antibodies is described in detail,taking monoclonal antibodies as an example. The antibody of the presentinvention may be a monoclonal antibody obtained by utilizing the cellfusion technology using myeloma cells. It can be produced via thefollowing steps, for instance. (1) Preparation of an immunogenicantigen, (2) immunization of an animal with the immunogenic antigen, (3)preparation of myeloma cells, (4) cell fusion between antibody producingcells and myeloma cells, (5) selection of hybridomas (fused cells) andcloning, and (6) monoclonal antibody production.

(1) The immunogenic antigen can be prepared in the following manner.While, as mentioned above, the native type cauxin polypeptide or afragment derived therefrom (which may include parts of domainpolypeptide, fragments, partial peptides and synthetic peptides andwhich may be a recombinant cauxin polypeptide) may be used as theantigen, it is also possible to use, as the antigen, an appropriateoligopeptide chemically synthesized based on the information about theamino acid sequence of cauxin as determined. As typical examples, theremay be mentioned peptides comprising at least five consecutive aminoacid residues occurring in the region selected from the group consistingof (1) the amino acid sequence shown under SEQ ID NO: 2 in the sequencelisting or the amino acid sequence of a partial domain(s) thereof (2)the amino acid sequence constituting the C terminal side domain out ofthe sequence shown under SEQ ID NO: 2 or a partial fragment thereof or(3) the amino acid sequence constituting the N terminal side domain outof the sequence shown under SEQ ID NO: 2 out of the sequence shown underSEQ ID NO: 2 in the sequence listing or a partial fragment thereof.

The antigen as such may be used in admixture with an appropriateadjuvant for immunizing an animal, or may be used in the form of animmunogenic conjugate. The antigen to be used as the immunogen may beone derived from cauxin by fragmentation or a synthetic polypeptidefragment obtained by chemical synthesis following polypeptide designingby selecting a characteristic sequence region based on the amino acidsequence of cauxin. It is also possible to bind that fragment to variouscarrier proteins via an appropriate condensing agent to give immunogenicconjugates such as hapten-protein conjugates and design monoclonalantibodies capable of reacting with specific sequences alone (orrecognizing specific sequences alone) using such conjugates. A cysteineresidue, for instance, may be added in advance to the polypeptidedesigned to thereby facilitate the preparation of immunogenicconjugates. In binding to carrier proteins, the carrier proteins can befirst activated. For such activation, an activating binding group isintroduced. The activating binding group includes (1) activated ester oractivating carboxyl groups, such as nitrophenyl ester, pentafluorophenylester, 1-benzotriazole ester, and N-succinimide ester groups, and (2)activating dithio groups, such as 2-pyridyldithio and like groups. Thecarrier proteins may include among others, keyhole lympet hemocyanin(KLH), bovine serum albumin (BSA), egg albumin, globulin, polypeptidessuch as polylysine, and bacterial cell components, for example BCG.

(2) Animals can be immunized with the immunogenic antigen in thefollowing manner. The immunization can be carried out by any of themethods known to those skilled in the art, for example according to themethods described in Shigeru Muramatsu et al. (ed.), Jikken SeibutsugakuKoza (Lectures on Experimental Biology) 14, Immunobiology, Maruzen,1985; Japanese Biochemical Society (ed.), Zoku Seikagaku Jikken Koza(Experiments in Biochemistry, Second Series) 5, Men'eki SeikagakuKenkyuho (Study Methods in Immunobiochemistry, Tokyo Kagaku Dozin, 1986;Japanese Biochemical Society (ed.), Shin Seikagaku Jikken Koza(Experiments in Biochemistry, Updated) 12, Molecular Immunology III,Antigens, Antibodies, and Complements, Tokyo Kagaku Dozin, 1992; and soon. Immunization can be attained by injecting the immunizing agent (ifnecessary together with an adjuvant) once or more times into mammals.Typically, the immunizing agent is subcutaneously or intraperitoneallyinjected, alone or together with an adjuvant, into mammals a pluralityof times. The immunizing agent includes the above-mentioned antigenpeptides or peptide fragments related thereto. The immunizing agent maybe used in the form of a conjugate with a protein (e.g. one of theabove-mentioned carrier proteins) known to be antigenic in the mammalsto be treated for immunization. The adjuvant includes, among others,Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, lipidA, liposomes, aluminum hydroxide, and silica. The immunization iscarried out using, for example, BALB/c and other mice, hamsters, orother appropriate animals. The dose of the antigen is about 1 to 400μg/animal in mice, for instance, and the antigen is generally injectedintraperitoneally or subcutaneously into the host animals and,thereafter, boosters are repeated about 2 to 10 times at intervals of 1to 4 weeks, preferably 1 to 2 weeks, by the intraperitoneal,subcutaneous, intravenous or intramuscular route. Not only BALB/c micebut also F1 mice from BALB/c mice and mice of another strain may be usedas the mice to be subjected to immunization. If necessary, an antibodytiter measuring system may be constructed so that the extent ofimmunization of animals can be confirmed by antibody titer measurements.The antibody of the present invention may be one obtained from an animalimmunized in the above manner, including an antiserum, polyclonalantibody, etc.

(3) Myeloma cells can be prepared in the following manner. Theimmortally growing strain (tumor cell strain) to be used in cell fusioncan be selected from among cell strains incapable of producingimmunoglobulins, for example P3-NS-1-Ag4-1 (NS-1, Eur. J. Immunol., 6:511–519, 1976), SP-2/0-Ag14 (SP-2, Nature, 276: 269–270, 1978), mousemyeloma MOPC-21 cell line-derived P3-X63-Ag8-U1 (P3U1, Curr. TopicsMicrobiol. Immunol., 81: 1–7, 1978), P3-X63-Ag8 (X63, Nature, 256:495–497, 1975), P3-X63-Ag8-653 (653, J. Immunol 123: 1548–1550, 1979)and the like. 8-Azaguanine-resistant mouse myeloma cell strains aresubcultured on a cell culture medium, such as Dulbecco's MEM (DMEM) orRPMI-1640 medium, supplemented with an antibiotic, such as penicillin oramikacin, fetal calf serum (FCS) and the like, and further supplementedwith 8-azaguanine (e.g. 5 to 45 μg/ml). A necessary number of cells canbe prepared by subculturing on a normal medium 2 to 5 days before cellfusion. The cell strain to be used may also be prepared by completelythawing the cryopreserved cell strain at about 37° C., washing three ormore times with a normal medium, such as RPMI-1640 medium, andcultivating on a normal medium until a required number of cells areobtained.

(4) The cell fusion between antibody-producing cells and myeloma cellscan be carried out in the following manner. The spleen of an animal, forexample a mouse, immunized in the above step (2) is excised 2 to 5 daysafter the final immunization, and a spleen cell suspension is preparedtherefrom. In addition to spleen cells, lymph node cells obtained fromvarious sites of the organism may also be used for cell fusion. Morespecifically, the cell fusion is carried out, for example, in anordinary nutrient culture fluid in the presence of a cell fusionpromoter. Usable as the culture fluid in the above cell fusion are suchmedia favorable for the growth of the above-mentioned myeloma cells asRPMI 1640 culture fluid and MEM culture fluid, and culture fluidscommonly used in this kind of cell culture. Auxiliary serum such asfetal calf serum (FCS) may further be used combinedly. The thus-obtainedspleen cell suspension and the myeloma cell line obtained in the abovestep (3) are placed in a cell culture medium, such as minimum essentialmedium (MEM), DMEM or RPMI-1640 medium, and a cell fusion promoter, forexample polyethylene glycol, is added. The cell fusion promoter may beany of those known in this field of art, and inactivated Sendai virus(HVJ; hemagglutinating virus of Japan) and the like may also bementioned as such.

Preferably, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added,polyethylene glycol species having a molecular weight of 1,000 to 8,000can be used, and polyethylene glycol species having a molecular weightof 1,000 to 4,000 are more preferably used. The concentration ofpolyethylene glycol in the fusion medium is preferably 30 to 60%, forinstance. If necessary, a small amount of such an auxiliary as dimethylsulfoxide may be added to thereby increase the fusion efficiency. Themixing ratio between immunocytes and myeloma cells, namely the ratiospleen cells (lymphocytes):myeloma cells to be used for fusion, may beselected arbitrarily and, for example, may be 1:1 to 20:1, morepreferably 4:1 to 10:1.

In cell fusion, the immunized cells and myeloma cells, in apredetermined mixing ratio, are thoroughly blended in the culture fluid,and a PEG solution (e.g. with an average molecular weight of about 1,000to 6,000) warmed at about 37° C. is added generally to a concentrationof 30 to 60% (w/v), and the mixture is stirred, whereby the desiredfused cells (hybridomas) are formed. Then, the procedure comprisingadding an appropriate culture fluid and removing the supernatant bycentrifugation is repeated to thereby remove the cell fusion agent andother components unfavorable for the growth of hybridomas.

The fusion reaction is carried out for 1 to 10 minutes and, then, a cellculture medium such as RPMI-1640 medium is added. It is also possible torepeat the fusion treatment a plurality of times. After the fusiontreatment, cells are separated by centrifugation and then transferred toa selective medium.

(5) Hybridomas (fused cells) can be selected and cloned in the followingmanner. The selective medium may include conventional selective culturefluids, such as FCS-containing MEM, RPMI-1640 medium or like mediacontaining hypoxanthine, aminopterine and thymidine, namely theso-called HAT media. In such a HAT medium as mentioned above, thecultivation is continued for a period (generally several days to severalweeks) sufficient for other cells (unfused cells) than the desiredhybridomas to die out. A method of selective medium exchange generallycomprises adding, on the next day, a volume equal to the volume of theportion distributed onto the culture plate and thereafter replacing ahalf of that volume with a fresh portion of the HAT medium at intervalsof 1 to 3 days. Appropriate modifications of this method may also beused. On the 8th to 16th days after fusion, the medium exchange may beconducted with the so-called HT medium, which is free of aminopterine,at intervals of 1 to 4 days. Mouse thymocytes, for instance, may be usedas feeder cells, and this is preferred in some instances.

Screening is performed by assaying, for the desired antibody, theculture supernatant in a well in which a vigorous hybridoma growth isseen in a measuring system for radioimmunoassay (RIA), enzymeimmunoassay (ELISA), fluorescent immunoassay (FIA), luminescentimmunoassay (LIA) or western blotting, for instance, or afluorescence-activated cell sorter (FACS), using the predeterminedfragment peptide as an antigen or using a labeled anti-mouse antibody.The hybridomas producing the desired antibody are subjected to cloning.The cloning can be carried out by picking up colonies in agar medium orby the limiting dilution method. The limiting dilution method is morepreferred. The cloning is preferably repeated a plurality of times. Thethus-produced monoclonal antibody producing hybridomas can besubcultured in an ordinary culture fluid and can be stored in liquidnitrogen for a long period of time.

(6) The monoclonal antibody production can be carried out in thefollowing manner. For obtained a monoclonal antibody from thecorresponding hybridomas, there may be employed, for example, a methodcomprising cultivating the hybridomas by a conventional method andrecovering the monoclonal antibody as the culture supernatant, or amethod comprising administering the hybridomas to a mammal compatibletherewith and allowing them to grow and recovering the antibody as theascitic fluid from the mammal. The former method is suited for obtaininga highly pure antibody, while the latter method is suited for massproduction of an antibody.

Thus, by cultivating the hybridoma strain obtained in an appropriategrowth medium, such as FCS-containing MEM or RPMI-1640 medium, it ispossible to obtained the desired monoclonal antibody from the culturesupernatant. For obtained the antibody in large quantities, the methodcomprising allowing the hybridomas to grow in the ascitic fluid may bementioned. In this case, hybridomas of each strain are transplanted intothe peritoneal cavity of a histocompatible animal isogenic to the animalserving as the origin of myeloma cells and allowed to grow, ortransplanted into a nude mouse, for instance, and allowed to grow. Themonoclonal antibody produced in the ascitic fluid in the animal can thenbe recovered. Prior to hybridoma transplantation, the animal may beintraperitoneally administered with a mineral oil, such as pristane(2,6,10,14-tetramethylpentadecane). After hybridoma growth followingthis treatment, the ascitic fluid can be collected. The ascitic fluidmay be used as the monoclonal antibody either as such or afterpurification by conventional methods, for example salting out by theammonium sulfate precipitation, for instance, gel filtration usingSephadex, ion exchange chromatography, electrophoresis, dialysis,ultrafiltration, affinity chromatography, high-performance liquidchromatography and/or the like. Preferably, the monoclonalantibody-containing ascitic fluid can be purified and isolated byammonium sulfate fractionation, followed by treatment with an anionexchange gel, such as DEAE-Sepharose, and an affinity column, such as aprotein A column. Especially preferred are affinity chromatography withan immobilized antigen or antigen fragment (e.g. synthetic peptide,recombinant antigen protein or peptide, site specifically recognized bythe antigen) column, affinity chromatography with an immobilized proteinA column, and hydroxyapatite chromatography, among others.

Further, transgenic mice or other organisms, for example other mammals,can be used in causing expression of antibodies to the immunogenpolypeptide product of the present invention.

It is also possible to determine the sequences of the antibodies thusobtained in large quantities and/or produce antibodies by the generecombination technology utilizing the nucleic acid sequence coding forthe antibody obtained from a hybridoma strain. The nucleic acid codingfor the monoclonal antibody can be isolated for sequencing by aconventional method, for example by using an oligonucleotide probecapable of specifically binding to the gene coding for the heavy chainor light chain of the mouse antibody. Once isolated, the DNA can beinserted into an expression vector for introduction into CHO, COS orlike host cells. The DNA can be modified, for example, by substituting asequence coding for a feline heavy chain or light chain constant regiondomain for the homogeneous murine sequence (Morrison et al., Proc. Natl.Acad. Sci. USA, 81: 6581, 1984). Thus, it is possible to prepare achimera antibody or hybrid antibody having the desired bindingspecificity. It is also possible to modify the antibody by applying atechnology of chemical protein synthesis, including the use of such acondensing agent as mentioned below, to give a chimera antibody orhybrid antibody.

Further, such antibody fragments as Fab, Fab′ and F(ab′)₂ obtained bytreatment of those antibodies with an enzyme such as trypsin, papain orpepsin, if necessary followed by reduction may also be used.

The antibodies can be used in any of the known assay methods, forexample competitive binding assay, direct and indirect sandwich assay,and immunoprecipitation (Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147–158 (CRC Press, Inc. 1987)). For conjugating theantibodies to a detectable atomic group, any of the methods known in therelevant field can be used and, thus, for example, mention may be madeof the methods described in David et al., Biochemistry, Vol. 13, pp.1014–1021 (1974); Pain et al., J. Immunol. Meth., 40: pp. 219–231(1981); and “Methods in Enzymology”, Vol. 184, pp. 138–163 (1990).Usable as the antibody to be labeled are an IgG fraction and, further,the specific binding portion Fab′ obtainable by reduction followingpepsin digestion. In these cases, the label includes enzymes(peroxidase, alkaline phosphatase or β-D-galactosidase, etc.), chemicalsubstances, fluorescent substances and radioisotopes, among others.

The detection and assay in the practice of the present invention can beperformed by immunostaining, for example tissue or cell staining, immuneelectron microscopy, immunochromatography, or immunoassay, for examplecompetitive immunoassay or noncompetitive immunoassay, andradioimmunoassay (RIA), FIA, LIA, EIA, ELISA and the like may also beused. B-F separation may be made, or assaying can be made without suchseparation. Preferred are RIA, EIA, FIA, LIA and, further, sandwichassay. In sandwich assay, for instance, the anti-cauxin polypeptideantibody of the present invention or the antibody to a cauxin-relatedpeptide fragment is used as one antibody, and the antibody to theC-terminal side residues of cauxin as the other, and one of them islabeled for rendering the same detectable (of course, other combinationsare also possible and appropriate designing can be made according to theintended purpose). The other antibody capable of recognizing the sameantigen is immobilized on a solid phase. The sample, labeled antibodyand immobilized antibody are subjected to incubation treatment accordingto need for the successive reactions and, after separating the unboundantibodies, the label is assayed. The quantity of the label as assayedis proportional to the quantity of the antigen, namely the cauxinpolypeptide antigen. This assay is called simultaneous sandwich assay,forward sandwich assay or reversed sandwich assay according to the orderof addition of the immobilized antibody and labeled antibody.

Washing, stirring, shaking, filtration and/or preliminary antigenextraction, for instance, may suitably be employed in the process ofassaying under specific conditions. Other assaying conditions such asspecific reagents, concentrations of buffer solutions and the like,temperature and incubation treatment time can be varied according to theantigen concentration in the specimen, the nature of the test sample andother factors. The one skilled in the art will be able to carry outassaying by appropriately selecting optimum and effective conditions foreach assay while using an ordinary method of experiment.

A large number of carriers are known for immobilizing antigens orantibodies and, appropriate ones may be selected from among them for usein the practice of the present invention. Various carriers are known tobe useful in antigen-antibody and like reactions and, of course,appropriate ones can be used for use in the practice of the presentinvention. Especially preferred for use are glass, for example activatedglass such as aminoalkylsilylated glass, porous glass, silica gel,silica-alumina, alumina, magnetized iron, magnetized alloys and otherinorganic materials, polyethylene, polypropylene, polyvinyl chloride,polyvinylidene fluoride, polyvinyl polymers, polyvinyl acetate,polycarbonates, polymethacrylates, polystyrene, styrene-butadienecopolymers, polyacrylamide, crosslinked polyacrylamide,styrene-methacrylate copolymers, polyglycidyl methacrylate,acrolein-ethylene glycol dimethacrylate copolymers and the like,crosslinked albumin, collagen, gelatin, dextran, agarose, crosslinkedagarose, cellulose, microcrystalline cellulose, carboxylmethylcellulose,cellulose acetate and other natural or modified cellulose, crosslinkeddextran, nylons and other polyamides, polyurethanes, polyepoxy resinsand other organic polymers, such polymers obtained by emulsionpolymerization, silicone rubbers and the like, cells, erythrocytes andthe like. If necessary, they may have a functional group introducedtherein using a silane coupling agent, for instance.

Furthermore, there may be mentioned filter paper, beads, tubes,cuvettes, inside walls of test vessels such as test tubes, titer plates,titer wells, microplates, glass cells, synthetic resin cells and cellsmade of some other synthetic material, and the surfaces of solidsubstances (bodies) such as glass rods, rods made of a syntheticmaterial, rods having a thickened or tapered end, rods having a roundprojection or flat projection at an end, and thin plate-like rods.

Antibodies can be bound to these carriers and, preferably, theanti-cauxin antibody (inclusive of antiserum or purified antibody) oranti-cauxin monoclonal antibody specifically reacting with the antigenobtained in accordance with the present invention can be bound to thosecarriers. The binding of the carrier with these components to beinvolved in the antigen-antibody reaction can be realized by physicalmeans such as adsorption, by chemical means using a condensing agent oran activated form, or by means utilizing a mutual chemical bindingreaction, for instance. As the label, there may be mentioned enzymes,enzyme substrates, enzyme inhibitors, prosthetic groups, coenzymes,enzyme precursors, apoenzymes, fluorescent substances, chromophores,chemoluminescent compounds, luminescent substances, chromogens, magneticsubstances, metal particles, for example gold colloid, nonmetallicelement particles, for example selenium colloid, radioactive substances,and so forth. The enzymes include dehydrogenases, reductases, oxidasesand other oxidation-reduction enzymes, transferases catalyzing thetransfer of an amino, carboxyl, methyl, acyl or phosphoryl group, forinstance, hydrolases hydrolyzing the ester, glycoside, ether or peptidebond, for instance, lyases, isomerases, ligases and so on. A pluralityof enzymes may be utilized in combination for detection purposes.

Enzymatic cycling, for instance, may also be utilized. As the isotopelabels of typical radioactive substances, there may be mentioned [³²P],[¹²⁵I], [¹³¹I], [³H], [¹⁴C], [³⁵S] and so forth. As typical enzymelabels, there may be mentioned horseradish peroxidase and otherperoxidases, Escherichia coli-derived β-D-galactosidase and othergalactosidases, malate dehydrogenase, glucose-6-phosphate dehydrogenase,glucose oxidase, glucoamylase, acetylcholine esterase, catalase, bovinesmall intestine-derived alkaline phosphatase, Escherichia coli-derivedalkaline phosphatase and other alkaline phosphatases, among others. Whenalkaline phosphatase is used, assaying can be made using anumbelliferone derivative such as 4-methylumbelliferyl phosphate, aphosphorylated phenol derivative such as nitrophenyl phosphate, anenzymatic cycling system utilizing NADP, a luciferin derivative, adioxetane derivative or a like substrate and measuring the resultingfluorescence or chemiluminescence. A luciferin-luciferase system mayalso be utilized. When catalase is used, oxygen is formed upon reactionwith hydrogen peroxide and that oxygen can also be detected by means ofan electrode, for instance. The electrode may be a glass electrode, anion-selective electrode with a hardly soluble salt membrane, aliquid-membrane electrode or a polymer membrane electrode, for instance.

A biotin label and an enzyme-labeled avidin (streptavidin) may besubstituted for the enzyme label. Thus, it is possible to suitablyemploy a sensitivity increasing method known in the art, for example theuse of such a biotin-avidin system or the use of a secondary antibodysuch as an antibody to anti-galectin antibody. It is also possible touse a plurality of different labels. In such case, is also becomespossible to carry out a plurality of measurements continuously ordiscontinuously, and simultaneously or separately. In the practice ofthe present invention, enzyme-reagent combinations may also be utilizedfor the formation of signals, for example the combinations of4-hydroxyphenylacetic acid, o-phenylenediamine (OPD),tetramethylbenzidine (TMB), 5-aminosalicylic acid, 3,3-diaminobenzidinetetrahydrochloride (DAB), 3-amino-9-ethylcarbazole (AEC), tyramine,lucigenin, luciferin and derivatives thereof, Pholad luciferin and thelike with horseradish peroxidase, the combinations of Lumigen PPD,(4-methyl)umbelliferyl phosphate, p-nitrophenol phosphate, phenolphosphate, bromochloroindolyl phosphate (BCIP), AMPAK™ (DAKO), AmpliQ™(DAKO) and the like with alkaline phosphatase, the combinations of4-methylumbelliferyl-β-D-galactoside or like umbelliferyl galactosides,o-nitrophenyl-β-D-galactoside or like nitrophenyl galactosides and thelike with β-D-galactosidase or glucose-6-phosphate dehydrogenase, andthe combination of ABTS with glucose oxidase, and use can be made ofthose compounds which can form hydroquinone, hydroxybenzoquinone,hydroxyanthraquinone and like quinol compounds, lipoic acid, glutathioneand like thiol compounds, phenol derivatives, ferrocene derivatives andthe like under the action of enzymes or the like.

The fluorescent substances or chemiluminescent compounds includefluorescein isothiocyanate (FITC), rhodamine derivatives such asrhodamine B isothiocyanate, tetramethylrhodamine isothiocyanate (RITC)and tetramethylrhodamine isothiocyanate isomer R (TRITC),7-amino-4-coumarin-3-acetic acid, dansyl chloride, dansyl fluoride,fluorescamine, phycobilin protein, acridinium salts, luciferin,luciferase, aequorin and like luminols, imidazole, oxalate esters, rareearth chelate compounds, and coumarin derivatives, among others. Fordetecting the resulting signals, inclusive of chemiluminescence andfluorescence, or the like, visual observation may be employed, or a perse known apparatus may also be used; thus, for example, afluorophotometer or a plate reader may be used. For detecting the signalemitted by a radioisotope, for instance, a per se known apparatus may beused; for example, a gamma counter or scintillation counter or the likemay also be used.

The labeling can be carried out utilizing the reaction between a thiolgroup and a maleimide group, the reaction between a pyridyl disulfidegroup and a thiol group or the reaction between an amino group and analdehyde group, for instance, and an appropriate method can be selectedfrom among the known methods, methods obvious to those skilled in theart and, further, modifications thereof.

Further, condensing agents which can be used in preparing theabove-mentioned immunogenic conjugates and condensing agents which canbe used in binding to carriers, and the like can be used. Suchcondensing agents include, among others, formaldehyde, glutaraldehyde,hexamethylene diisocyanate, hexamethylene diisothiocyanate,N,N′-polymethylenebis-iodoacetamide, N,N′-ethylenebismaleimide, ethyleneglycol bissuccinimidyl succinate, bisdiazobenzidine,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, succinimidyl3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),N-sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,N-succinimidyl (4-iodoacetyl)aminobenzoate, N-succinimidyl4-(1-maleimidophenyl)butyrate, N-(ε-maleimidocaproyloxy)succinimide(EMCS), iminothiolane, S-acetylmercaptosuccinic acid anhydride, methyl3-(4′-dithiopyridyl)propionimidate, methyl 4-mercaptobutyrylimidate,methyl 3-mercaptopropionimidate, N-succinimidyl S-acetylmercaptoacetate,etc.

According to the assay method of the present invention, a substance tobe assayed can be reacted successively or simultaneously with a labeledantibody reagent such as an antiserum, purified antibody or monoclonalantibody labeled with an enzyme or the like, and an antibody bound to acarrier. The order of addition of the reagents may vary according to thetype of the carrier system selected. When a sensitized plastic bead orlike bead is used, the assay can be performed by first placing thelabeled antibody reagent such as a labeled antiserum, purified antibodyor monoclonal antibody, together with a test sample containing asubstance to be assayed, in an appropriate test tube, and then addingthe sensitized plastic or like bead.

In the assay method of the present invention, an immunological assaymethod is used and, on that occasion, the solid carrier to be used canbe arbitrarily selected from among various carriers differing inmaterial and shape, for example balls, microplates, sticks, minuteparticles and test tubes made of a material on which proteins such asantibodies can be readily adsorbed, for example polystyrene, apolycarbonate, polypropylene or a polyvinyl.

The assay can be carried out in an appropriate buffer solution system sothat an optimum pH, for example a pH of about 4 to about 9, can bemaintained. Especially suited buffers are, for example, acetate,citrate, phosphate, Tris, triethanolamine, borate, glycine, carbonate,Tris-hydrochloride and veronal buffers. The buffers may be used incombination in an arbitrary mixing ratio. The antigen-antibody reactionis preferably carried out at a temperature between about 0° C. and about60° C.

The incubation treatment of the antibody reagent such as an antiserum,purified antibody or monoclonal antibody labeled with an enzyme or thelike, the antibody reagent bound to a carrier and, further, thesubstance to be assayed may be carried out until an equilibrium isarrived at. However, the antigen-antibody reaction can be terminated,after restricted incubation treatment, by separating the solid phasefrom the liquid phase at a time by far earlier than the time when thereaction arrives at an equilibrium, and the extent of occurrence of thelabel such as an enzyme in the liquid phase or in the solid phase can bemeasured. The assay procedure can be performed using an automatedmeasurement apparatus, and the indicator signal generated uponconversion of the substrate under the action of the enzyme can bedetected and measured using a luminescence detector or photo detector orthe like. In carrying out the antigen-antibody reaction, appropriatemeasures can be taken so that the reagents to be used, the substance tobe assayed and, further the label such as an enzyme may be stabilized orthe antigen-antibody reaction itself may be stabilized.

Further, a protein, a stabilizing agent, such a surfactant as mentionedbelow, a chelating agent and/or the like may be added to the incubationsolution so that nonspecific reactions may be eliminated, inhibitoryinfluences may be reduced and/or the assaying reaction may be activated.Ethylenediaminetetraacetate salts (EDTA) are more preferred as thechelating agent. The blocking treatment commonly employed in therelevant field or known to those skilled in the art may be carried outfor preventing nonspecific binding reactions. Thus, for example, normalmammalian or other sera or serum proteins, albumin, hemoglobin,ovalbumin (OVA), skimmed milk, milk fermentation substances, collagen,gelatin can be used for the treatment. So long as the purpose is toprevent nonspecific binding reactions, those methods can be used withoutany particular restriction. Further, the samples, solid phases andothers can be washed with an appropriate solution selected from amongthe buffer solutions systems mentioned above and saline and, further, asurfactant selected from the group consisting of Tween 20 (trademark),Tween 80 (trademark), NP-40 (trademark), Triton X100 (trademark), Briji(trademark) and like nonionic surfactants, CHAPS and like amphotericsurfactants, cationic surfactants and anionic surfactants, among others,may be added to the solution.

The sample to be assayed according to the assay of the present inventionincludes various forms of solutions, colloidal solutions, nonfluidsamples, etc. Preferred are, however, living body-derived samples, forexample organs and tissues such as kidneys, urine, other body fluids,cell culture fluids, tissue culture fluids, tissue homogenates, biopsysamples, cells, and the like.

According to the present invention, a feline urinary protein testing kitin which an anti-cauxin antibody is used is preferably provided. Thebasic constitution of the kit may be similar to those of human pregnancytesting kits as disclosed in JP-A-H09-171019 and JP-A-H10-132817, forinstance, and of simplified methods currently in general use. Forexample, the testing kit is a cauxin detection kit characterized in thatit is composed of (a) a sample application site, (b) a labeledantibody-containing site, (c) an antigen detection site and (d) areaction completion judging site, as disposed in that order on a carrieror support enabling substances to move in a wet condition, wherein (i)the labeled antibody-containing site contains a labeled anti-cauxinantibody (labeled antibody) which is capable of migrating on the carrierto the antigen detection site and then to the reaction completionjudging site in a wet condition, (ii) an immobilized anti-cauxinantibody (immobilized antibody) is positioned on the detection site, and(iii) a site where an antibody (second antibody) to the antibody used asthe labeled antibody is immobilized is formed on the reaction completionjudging site, and wherein when a sample is applied to the sampleapplication site, the sample is thus allowed to migrate on the carrier,elute the labeled antibody and pass through the immobilized antibody onthe antigen detection site and through the second antibody site on thereaction completion judging site, for detecting cauxin in the sample, aswell as the respective reagents to be used in the detection kit.

In a typical example, gold colloid immunochromatography, for instance,may be mentioned. The gold colloid immunochromatographic methodcomprises, for example, (1) preparing a gold colloid-labeled antibody bybinding an appropriate amount of an anti-cauxin antibody to a goldcolloid solution; (2) preparing an oblong strip-like filter paper onwhich a sample padding site for dropping a liquid sample, a gold colloidphase where the gold colloid-labeled antibody is positioned, a trappingsite with the anti-cauxin antibody immobilized thereon, and an absorbentpad for absorbing the reaction mixture, with the four elements beingdisposed in line; and (3) dropping a cauxin-containing feline urinesample onto the sample pad, whereupon the sample migrates to the goldcolloid phase owing to the chromatographic phenomenon and urinary cauxinis bound to the gold colloid-labeled antibody; (4) the gold colloid withcauxin bound to the surface thereof then further diffuses to thetrapping site where the gold colloid is immobilized by the antibody onthe trapping site; and (5) upon flocculation of the gold colloid on thetrapping site, the site visually turns colored red purple. If cauxin isabsent in the urine sample, the gold colloid passes through the trappingsite and migrates to the absorbent pad, so that no coloration can beobserved. When the urinary concentration of cauxin is high, the amountof the gold colloid flocculate increases, hence the coloration becomesintense (or the colored area extends). Thus, quantitative assayingbecomes possible.

The carrier for enabling substances to migrate in a wet condition may beselected from among the carriers mentioned hereinabove, for examplethose known as porous carriers. The carrier can be prepared from anarbitrary material, such as a hygroscopic material, a porous material ora fibrous material. The material is preferably a porous one, and theporosity may be unidirectional or multidirectional. The term“unidirectional” as used herein may mean that all fibers or pores arelined up in parallel to the axis of the carrier, and the term“multidirectional” may mean omnidirectional, namely that fibers or poresshow no particular orientational tendency with respect to liquidpermeation or penetration. Preferred examples of the carrier arepolypropylene, polyethylene, polyvinylidene fluoride, ethylene-vinylacetate copolymers, polyacrylonitrile, polytetrafluoroethylene and likeplastics, paper, filter paper, nitrocellulose and other variouscellulosic materials. The label for the labeled antibody may be selectedfrom among the labels mentioned hereinabove, and those giving signalswhich are directly visible by the eye, for example gold colloid, pigmentor dye sols, and colored latexes, are used with advantage. The labeledantibody can be applied to the carrier by injecting into the carrier orapplying to the surface or layering on the surface using a microsyringeor microinjector, for instance. The technique for antibodyimmobilization may be selected from among those mentioned hereinabove.

In applying various analytical and assaying method, inclusive of theseindividual immunological assay methods, to the assay method of thepresent invention, it is not necessary to select any particularconditions or particular procedure. It is only necessary to construct anassay system relating to the target substance according to the presentinvention or a substance substantially equivalent in activity theretowhile adding ordinary technical considerations of the one skilled in theart to general conditions and procedure for each analytical/assaymethod.

Epitope mapping can also be made using the anti-cauxin antibody of thepresent invention, in particular a monoclonal antibody. When antibodiesrecognizing respective epitopes are used, each of cauxin and relatedpeptide fragments and the like can be detected and/or assayed.

As mentioned hereinabove, the feline nephropathy diagnosing/detectingagent comprising an anti-cauxin antibody obtained as a polyclonalantibody (inclusive of antiserum) or monoclonal antibody can be used inimmunohistologically analyzing the cauxin levels using, as samples,cells extracted from tissues such as kidneys. Thus, feline nephropathycan be detected and diagnosed by analyzing the cauxin level in targetcells.

Here, the feline nephropathy diagnosing agent may comprise ananti-cauxin antibody as an active ingredient. The term “comprising as anactive ingredient” means that the anti-cauxin antibody is contained at asufficient concentration to detect feline nephropathy. The recognitionsite of the anti-cauxin antibody is not limited to the epitope but maybe at any region of the cauxin protein.

The term “cauxin” as used herein may mean not only cauxin shown underSEQ ID NO: 2 in the sequence listing but also natural mutants of cauxin,mutants derived therefrom by artificial mutation (namely, deletion,addition, modification or insertion, for instance, of one or more aminoacid residues) and partial domains or partial peptide fragments.

As typical anti-cauxin antibodies, there may be mentioned antibodiesbinding to all such cauxin species, antibodies specific to theC-terminal side domain of cauxin or fragment peptides thereof, andantibodies specific to the N-terminal side domain of cauxin or fragmentpeptides thereof, for instance.

Advantageous for utilization in the cauxin assaying system are suchprotein assaying systems as immunostaining (METHODS, 24, 289–296 (2001);J. Immunol Methods, 47 (2), 129–144 (1981); ibid., 150 (1–2), 5–21,23–32 & 151–158 (1992); Cancer J., 7 (1), 24–31 (2001), etc.) and immuneelectron microscopy (Mol. Biotechnol., 7 (2), 145–151 (1997); J.Electron Microsc. Tech., 19 (1), 57–63 & 64–79 (1991); ibid., 19 (3),305–315 (1991), etc.) and such expressed gene assaying systems as insitu hybridization in the case of tissue samples, for instance, suchprotein assaying systems as EIA, RIA, FIA, LIA, western blotting (J.Electron Microsc. (Tokyo), 45 (2), 119–127 (1996); Methods Biochem.Anal., 33, 1–58 (1988); Methods Enzymol., 271, 177–203 (1996); ibid.,305, 333–345 (2000); J. Immunol Methods, 152 (2), 227–236 (1992); ibid.,170 (2), 177–184 (1994); ibid., 195 (1–2) 149–152 (1996); YoshiyukiKuchino et al. (ed.), “Idenshi/Tanpakushitsu, Jikken Sosa(Genes/Proteins, Experimental Procedures): Blotting”, Kabushiki KaishaSoft Science, published Nov. 10, 1987, etc.) and such expressed geneassaying systems as northern blotting, dot blot, RNase protection assay,RT-PCR (reverse transcription polymerase chain reaction), Real-Time PCR(Clinical Chemistry, 46: 11, 1738–1743 (2000) for tissue extracts, andsuch protein assaying systems as EIA, RIA, FIA, LIA, and westernblotting for blood, body fluid and like sample. For the anti-cauxinantibody assaying systems, such protein assaying systems as EIA, RIA,FIA, LIA, and western blotting can advantageously be utilized for blood,body fluid and like samples.

In EIA systems, in the case of competitive method, for instance, theanti-cauxin antibody is used as an immobilized antibody and a labeledantigen and an unlabeled antigen (cauxin or a fragment peptide thereofmay be mentioned as the antigen) are used and, in the noncompetitivemethod, for example the sandwich method, an immobilized anti-cauxinantibody or a labeled anti-cauxin antibody can be utilized or theanti-cauxin may be directly labeled or an antibody to the anti-cauxinantibody may be labeled without immobilization or with immobilization.As the sensitivity increasing method, there may be mentioned theutilization, in the combination with a non-enzyme-labeled primaryantibody, of a macromolecular polymer and an enzyme and the primaryantibody (application of Envision reagent: Enhanced polymer one-stepstaining (EPOS)) and, in the combination with a non-enzyme-labeledsecondary antibody, the combination of an enzyme and an anti-enzymeantibody complex as in the PAP (peroxidase-antiperoxidase) technique,the combination of a biotin-labeled secondary antibody and abiotin-labeled enzyme-avidin complex as in the SABC (avidin-biotinylatedperoxidase complex) method, the combination of a biotin-labeledsecondary antibody and a biotin-labeled enzyme-streptavidin complex asin the ABC (streptavidin-biotin complex) method or the LSAB (labeledstreptavidin-biotin) method, the combination of SABC, a biotin-labeledtyramide and an enzyme-labeled streptavidin as in the CSA (catalyzedsignal amplification) method, and the use of a macromolecular polymerlabeled with a secondary antibody and an enzyme, among others.

For the particulars of these general technological means, reviewarticles, monographs and so on may be referred to [for example, HiroshiIrie (ed.), “Radioimmunoassay”, Kodansha, published 1974; Hiroshi Irie(ed.), “Radioimmunoassay, Volume Two”, Kodansha, published 1979; EijiIshikawa et al. (ed.), “Koso Men'eki Sokuteiho (Enzyme Immunoassay)”,Igaku Shoin, published 1978; Eiji Ishikawa et al. (ed.), “Koso Men'ekiSokuteiho” (second edition), Igaku Shoin, published 1982; Eiji Ishikawaet al. (ed.), “Koso Men'eki Sokuteiho” (third edition), Igaku Shoin,published 1987; H. V. Vunakis et al. (ed.), “Methods in Enzymology”,Vol. 70 (Immunological Techniques, Part A), Academic Press, New York(1980); J. J. Langone et al. (ed.), “Methods in Enzymology”, Vol. 73(Immunological Techniques, Part B), Academic Press, New York (1981); J.J. Langone et al. (ed.), “Methods in Enzymology”, Vol. 74 (ImmunologicalTechniques, Part C), Academic Press, New York (1981); J. J. Langone etal. (ed.), “Methods in Enzymology”, Vol. 84 (Immunological Techniques,Part D: Selected Immunoassays), Academic Press, New York (1982); J. J.Langone et al. (ed.), “Methods in Enzymology”, Vol. 92 (ImmunologicalTechniques, Part E: Monoclonal Antibodies and General ImmunoassayMethods), Academic Press, New York (1983); J. J. Langone et al. (ed.),“Methods in Enzymology”, Vol. 121 (Immunological Techniques, Part I:Hybridoma Technology and Monoclonal Antibodies), Academic Press, NewYork (1986); J. J. Langone et al. (ed.), “Methods in Enzymology”, Vol.178 (Antibodies, Antigens, and Molecular Mimicry), Academic Press, NewYork (1989); M. Wilchek et al. (ed.), “Methods in Enzymology”, Vol. 184(Avidin-Biotin Technology), Academic Press, New York (1990); J. J.Langone et al. (ed.), “Methods in Enzymology”, Vol. 203 (MolecularDesign and Modeling: Concepts and Applications, Part B: Antibodies andAntigens, Nucleic Acids, Polysaccharides, and Drugs), Academic Press,New York (1991); and references cited therein (the descriptions thereinare incorporated herein by reference)].

In another aspect, the present invention provides a feline nephropathydetecting/diagnosing agent characterized by containing a nucleic acidcapable of hybridizing with a nucleic acid coding for cauxin or aconstituent domain thereof, a feline nephropathy detecting method usingthe same, and a system to be utilized therefor. As the hybridizingnucleic acid, there may be mentioned, for example, probes and primers.Probes capable of hybridizing with a cauxin gene or a product thereofcan be utilized without restriction provided that the intended purposecan be attained. The above nucleic acid can be obtained according to the“gene manipulation technology” mentioned hereinabove. For example, itcan be obtained with ease by designing a plurality of primers utilizingthe information on the nucleotide base sequence shown under SEQ ID NO: 3in the sequence listing, synthesizing them and carrying out the PCR(polymerase chain reaction) using them, for instance. The primers can besynthesized by the methods known in the art, typically by thephosphodiester method, phosphotriester method, phosphoamidite method andlike methods. For example, they can be synthesized using an automatedDNA synthesizer, for example a model 381A DNA synthesizer (AppliedBiosystems). PCR is carried out using a cDNA library, together with asense primer and an antisense primer, whereby cDNA can be amplified. Thenucleic acid obtained can be used as a specific hybridization probe. Forlabeling the probe with a radioisotope, for instance, a commerciallabeling kit, such as a random prime DNA labeling kit (BoehringerMannheim), can be used. The probe DNA can be labeled with [α-³²P]dCTP(Amersham) or the like using a random priming kit (Pharmacia LKB,Uppsala), for instance, whereby a radioactive probe can be obtained. Thelabel of the probe may be any of those known in the relevant field ofart and can be adequately selected from among the labels mentionedhereinabove referring to the antibodies.

The hybridization is carried out by transferring the sample holding orcarrying the DNA in question to a membrane, such as a nylon filter and,after denaturation, immobilization, washing and/or like treatment, ifnecessary, reacting the sample transferred to the membrane with thelabeled probe DNA fragment (if necessary denatured) in a hybridizationbuffer. The hybridization treatment is generally carried out at about35° C. to about 80° C., more preferably at about 50° C. to about 65° C.,for about 15 minutes to about 36 hours, more preferably for about 1 hourto about 24 hours. Optimum conditions can properly be selected. Thus,for example, the hybridization treatment is carried out at about 55° C.for about 18 hours. The hybridization buffer can be selected from amongthose commonly used in the relevant field. Thus, for example, Rapidhybridization buffer (Amersham) can be used. As the denaturationtreatment of the membrane after DNA transfer, there may be mentioned themethod using an alkaline denaturing solution, and this treatment ispreferably followed by treatment with a neutralizing solution of abuffer solution. The immobilization treatment of the membrane is carriedout by baking generally at about 40° C. to about 100° C., morepreferably at about 70° C. to about 90° C., for about 15 minutes toabout 24 hours, more preferably for about 1 hour to about 4 hours.Optimum conditions can properly be selected. Thus, for example, theimmobilization is realized by baking the filter at about 80° C. forabout 2 hours. The washing treatment of the transferred DNA-carryingmembrane can be performed by washing in a washing solution commonly usedin the relevant field, for example with 50 mM Tris-HCl buffer, pH 8.0,containing 1 M NaCl, 1 mM EDTA and 0.1% sodium dodecyl sulfate (SDS).The membrane for receiving transferred DNA can be selected from amongthose generally used in the relevant field, and a nylon filter or thelike, for instance, may be mentioned.

The above alkaline denaturing solution, neutralizing solution and buffersolution can be selected respectively from among those generally used inthe relevant field. Thus, the alkaline denaturing solution may be, forexample, a solution containing 0.5 M NaOH and 1.5 M NaCl, theneutralizing solution may be, for example, 0.5 M Tris-HCl buffer, pH8.0, containing 1.5 M NaCl, and the buffer solution may be, for example,2×SSPE (0.36 M NaCl, 20 mM NaH₂PO₄ and 2 mM EDTA) or the like. Forpreventing nonspecific hybridization reactions, the membrane aftertransfer is preferably subjected to prehybridization treatment accordingto need prior to the hybridization treatment. This prehybridizationtreatment can be carried out, for example, by immersing the membrane ina prehybridization solution [50% formamide, 5× Denhardt's solution (0.2%bovine serum albumin, 0.2% polyvinylpyrrolidone), 5×SSPE, 0.1% SDS, 100μg/ml heat-denatured salmon sperm DNA] or the like, and allowingreactions to proceed at about 35° C. to about 50° C., preferably atabout 42° C., for about 4 to about 24 hours, preferably for about 6 toabout 8 hours. As for such conditions, the one skilled in the art willbe able to selected more preferred conditions after appropriatelyrepeated experiments. The labeled probe DNA fragment to be used forhybridization can be denatured, for example, by heating at about 70° C.to about 100° C., preferably at about 100° C., for about 1 minute toabout 60 minutes, preferably for about 5 minutes. The hybridization canbe carried out by a per se known method or a modification thereof. Theterm “stringent conditions” as used herein refers, for example, to suchconditions as a sodium concentration of about 15 to about 50 mM,preferably about 19 to about 40 mM, more preferably about 19 to about 20mM, and a temperature of about 35 to about 85° C., preferably about 50to about 70° C., more preferably about 60 to about 65° C.

After completion of the hybridization, the filter is sufficiently washedto remove the residual labeled probe other than the labeled probe DNAfragment that has participated in the hybridization reaction. The filtercan be washed with a washing solution selected from among thosegenerally used in the relevant field of art and, for example, the filtercan be washed with 0.5×SSC (0.15 M NaCl, 15 mM citric acid) solutioncontaining 0.1% SDS, or the like.

The site of hybridization can be detected typically by autoradiography.Any other technique properly selected from among the techniques used inthe relevant field may also be used.

Feline nephropathy can be detected by detecting/assaying the cauxinexpression gene (including DNA such as cDNA and RNA such as mRNA) by thetechniques known for detecting/assaying the expression of a specificgene in the relevant field according to the above-described “generecombination technology”, for example by in situ hybridization,northern blotting, dot blot, RNase protection assay, RT-PCR, Real-TimePCR (Journal of Molecular Endocrinology, 25, 169–193 (2000) andreferences cited therein), or DNA array analysis (Mark Shena (ed.),“Microarray Biochip Technology”, Eaton Publishing (March 2000). Thecauxin expression gene assay systems utilizing such techniques, and thereagents, techniques and processes to be utilized therein, for instance,are all included in the feline nephropathy detecting/diagnosing agent,feline nephropathy detecting/diagnosing method and system thereforaccording to the present invention. The in situ hybridization mentionedabove may include non-RI in situ hybridization, for instance, and thedirect method and indirect method, for instance, may be includedtherein. The direct method uses, for example, a detectable molecule(reporter) directly bound to the nucleic acid probe, while the indirectmethod uses, for example, an antibody to the reporter molecule forattaining signal amplification. The oligonucleotide in the nucleic acidprobe contains functional groups (e.g. primary amino group, SH group,etc.) as introduced therein, and a hapten, fluorescent dye or enzyme,for instance, may be bound to such a functional group. As typicalexamples of the label of the nucleic acid probe, there may be mentioneddigoxigenin (DIG), biotin, fluorescein and the like. The label, however,may be selected from among the labels described hereinabove referring tothe antibodies, and multiple labeling may also be employed and, further,labeled antibodies may also be utilized. The method of labeling thenucleic acid probe can be properly selected from among the methods knownin the relevant field and, thus, for example, there may be mentionedrandom priming, nick translation, DNA amplification by PCR,labeling/tailing, and in vitro transcription, among others. Forobserving the treated sample, an appropriate method can be selected fromamong those methods known in the art. For example, a dark fieldmicroscope, phase contrast microscope, reflection contrast microscope,fluorescence microscope, digital imaging microscope, or electronmicroscope can be used. Further, flow cytometry or the like may also beemployed.

In accordance with the present invention, cauxin and the cauxinexpression gene can be used as markers for feline nephropathy, andfeline nephropathy detecting/diagnosing agents, agents for detectionand/or measurement of such a disease, feline nephropathydetecting/diagnosing methods or abnormality detecting and/or measuringmethods and, further, reagent kits or systems for such detection and/ormeasurement can be prepared, established or constructed in various formsutilizing such markers. Furthermore, after treatment of felinenephropathy, they can be utilized also for making a prognosis in theform of disease detecting and/or measuring agents, disease detectingand/or measuring methods and, further, disease detection and/ormeasuring reagent sets or systems, with feline nephropathy as thetarget. They can be expected to show good functions and effects inprognosis as well.

The term “marker” as used herein may indicate that it makes it possibleto recognize or identify the “feline nephropathy (cat kidney diseases)”or “feline renal abnormality” and, further, may indicate that it canfunction as a measure of the level of the “disease” in question and theseverity of “symptoms”. It is to be considered that the term denotes thefunctions mentioned above according to the presence or absence of themarker and/or the difference in the quantity thereof.

By using a test kit with which feline urinary cauxin can be assayedusing an anti-cauxin antibody according to the present invention, theowner can check his/her family cat for health condition in a simplemanner without visiting a veterinarian. Further, it becomes possible tofind out feline nephropathy before the disease becomes incurable,whereby the life of the family cat can be prolonged. When used by aveterinarian, the kit can give an indicator which can be used in judgingthe extent of progress of nephropathy and in making a prognosis.

In the present specification and drawings, the terms used are thoseaccording to the IUPAC-IUB Commission on Biochemical Nomenclature orconventionally used in the relevant field of art.

EXAMPLES

The following examples illustrate the present invention specifically.These examples are, however, by no means limitative of the scope of thepresent invention. It is to be understood that various modes ofembodiment are possible based on the spirit and ideas disclosed herein.Unless otherwise specified, all the examples were carried out, or can becarried out, using standard techniques which are well known andconventional to those skilled in the art.

In the following examples, unless otherwise pointed out, the specificprocedures and treatment conditions employed are as described in thefollowing references: as regards DNA cloning, J. Sambrook, E. F. Fritch& T. Maniatis, “Molecular Cloning”, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989) and D. M. Glover et al. ed.,“DNA Cloning”, 2nd ed., Vol. 1 to Vol. 4, (The Practical ApproachSeries), IRL Press, Oxford University Press (1995); as regards PCR, inparticular, H. A. Erlich ed., PCR Technology, Stockton Press, 1989; D.M. Glover et al. ed., “DNA Cloning”, 2nd ed., Vol. 1, (The PracticalApproach Series), IRL Press, Oxford University Press (1995) and M. A.Innis et al. ed., “PCR Protocols”, Academic Press, New York (1990). Whencommercial reagents or kits were used, the protocols attached theretowere referred to and the chemicals or reagents attached thereto wereused.

Example 1

(1) Search for and Analysis of a Feline Urinary Protein

(a) For analyzing feline urinary proteins, urine samples were collectedfrom adult cats, kittens and cats with nephropathy and subjected toSDS-PAGE under nonreducing conditions.

Thus, a SDS sample buffer was added to 10 μl of urine from each cat(animal of the family Felidae), and the mixture was boiled for 3 minutesand then subjected to SDS-polyacrylamide electrophoresis (SDS-PAGE)under nonreducing conditions using a polyacrylamide gel (12%concentration) according to the method of Laemmli. Afterelectrophoresis, the gel was stained with a Coomassie Brilliant BlueR250 staining solution.

The results are shown in FIG. 1. In the normal healthy adult cat-derivedurine samples, a protein having a molecular weight of 70 k was foundexcreted in high concentrations, irrespective of sex. This protein wasnot detected in kitten-derived urine samples, while the urinaryexcretion of the protein was found markedly decreased in cats withnephropathy. This protein was named “cauxin”.

(b) For analyzing urinary proteins in animals belong to the familyFelidae, urine samples were collected from animals of the subfamilyFelinae g house cat group, subfamily Felinae wildcat group, subfamilyPantherinae or subfamily Acinonvchinae, and subjected to SDS-PAGE undernonreducing conditions in the same manner as in (a) above.

The results are shown in FIG. 2. In bobcat, serval and lynx, acauxin-like protein of 70 kDa was detected at the same position as theurinary protein (cauxin) identified as described above.

(2) Cloning and Analysis of the Gene Coding for Urinary Protein Cauxin

(a) Cloning of Cauxin cDNA

Cauxin purified from feline urine was analyzed for the N-terminal aminoacid sequence on a protein sequencer (model PPSQ-21, Shimadzu) and,after lysylendopeptidase treatment, the products were analyzed for 7internal partial amino acid sequences. Based on these results, PCRprimers were designed and prepared. RT-PCR was carried out usingmessenger RNA extracted from the feline kidney as a template. Aprobe wasprepared by labeling the amplified PCR product using the Alkphos directlabeling kit (Amersham Pharmacia Biotech). Then, a feline kidney cDNAlibrary constructed by using the SMART cDNA Library Construction Kit(Clontech) was screened using the above-mentioned probe, and the cauxincDNA was cloned and analyzed for nucleotide base sequence using a DNAsequencer (model ABI PRISM 310 DNA sequencer, Perkin-Elmer). Based onthe results, the full-length amino acid sequence of cauxin wasdetermined.

(b) Analysis of the Cauxin DNA Sequence and Amino Acid Sequence

The nucleotide base sequence of the cauxin DNA cloned as mentioned abovewas subjected to homology searching using the GenBank™ databases BLASTXand FASTA. Amino acid sequence comparison with carboxylesterase was madeusing the gene analysis software GENETYX-MAC. The DNA sequence codingfor cauxin is shown in the sequence listing under SEQ ID NO: 1, theamino acid sequence of cauxin in the sequence listing under SEQ ID NO:2, and the sequence of the cloned DNA coding for cauxin in the sequencelisting under SEQ ID NO: 3. In FIG. 3, there is shown the primarystructure of cauxin, in comparison with carboxylesterase. In FIG. 4,there is shown the C-terminal amino acid sequence alignment of cauxin,in comparison with carboxylesterases obtained from rat kidney, ratliver, swine liver and human liver. The C terminus of cauxin was lackingin endoplasmic reticulum retention signal (HXEL).

Example 2

(1) Production of a Polyclonal Antibody Against the Urinary ProteinCauxin

A cauxin C-terminal amino acid sequence composed of 20 residues wasprepared on a peptide synthesizer (model PPSM-8, Shimadzu) and mixedwith Freund's complete adjuvant, and the mixture was used to immunize arabbit. A cauxin-specific antibody was thus prepared.

(2) Analysis of Tissue Distribution of the Urinary Protein Cauxin

(a) Western Blotting

Using 1 μg each of the proteins extracted from feline serum, liver,kidney and pancreas, trials for cauxin detection were made with westernblotting. The protein (1 μg) extracted feline urine or the protein (5μg) extracted from each of feline kidney, liver and pancreas wasdeveloped by SDS-PAGE, followed by western blotting onto a membrane. Themembrane was blocked with 1% polyvinylpyrrolidone in TBS-T (10 mMTris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.4) and then treated with an8000-fold dilution of cauxin antiserum, a 10000-fold dilution ofanti-rabbit IgG-HRP (BioRad) and an ECL kit (Amersham PharmaciaBiotech), followed by detection using an X-ray film (FIG. 5). From FIG.5, it was confirmed that cauxin is localized in kidneys alone.

(b) Northern Blotting

RNA was extracted from each of various cat organs by the AGPC method.Each total RNA (2 μg) was developed on a 1% agarose gel supplementedwith paraformaldehyde, followed by northern blotting onto a membrane.After hybridization with the probe, the membrane was washed twice with1×SSC, 0.1% SDS at 65° C. for 15 minutes and further washed twice with0.1×SSC, 0.1% SDS at 65° C. for 15 minutes and, after treatment withCDP-star (Amersham Pharmacia Biotech), trials for detection were madeusing an X-ray film (FIG. 6). From FIG. 6, it was confirmed that cauxinis expressed in kidneys alone. For probe preparation, the cauxin RNA wasproduced from the full-length cauxin cDNA by in vitro transcription andthe RNA was labeled using a DIG lebeling kit (Roche Diagnostics).

Example 3

(1) Cauxin Expression in Feline Kidney and Analysis of the Site ofLocalization

(a) In Situ Hybridization

A feline kidney was fixed with 4% paraformaldehyde, a paraffin-embeddedsection was then prepared. For probe preparation, the cauxin RNA wasproduced from the full-length cauxin cDNA by in vitro transcription andthe RNA was labeled using a DIG lebeling kit (Roche Diagnostics). Afterhybridization with the probe on the paraffin section, the section wasreacted with alkaline phosphatase-conjugated anti-DIG antibodies (RocheDiagnostics) and the cauxin mRNA was detected using Nitro bluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics).

(b) Immunohistochemistry

After blocking with 10% swine serum (DAKO), the paraffin-embeddedsection was reacted with a 500-fold dilution of cauxin antiserumovernight at 4° C. After washing three times with Tween-PBS, the sectionwas reacted with anti-rabbit IgG swine serum (DAKO) at 37° C. for 1 hourand further treated with 0.02% diaminobenzidine tetrahydrochloride and0.005% H₂O₂ for detection of cauxin.

(c) Results

Cauxin was found localized in distal tubule epithelial cells in thecorticomedullar transitional zone in the adult cat kidney. FIG. 7 showsthe kidney of an adult cat. FIG. 8A shows the in situ hybridization(×50) patterns of the slanting line area shown in FIG. 7, and FIG. 8Bshows the immunohistochemical staining (×50) patterns of the slantingline area shown in FIG. 7. FIG. 9C shows the in situ hybridization(×100) patterns of a corticomedullar transitional zone of an adult catkidney, FIG. 9D is an enlargement (in situ hybridization (×400)) of thesite shown in FIG. 9C, and FIG. 9E shows the in situ hybridization(×200) patterns of a corticomedullar transitional zone of an adult catkidney. FIG. 10 shows the immunohistochemical staining (×100) patternsof a corticomedullar transitional zone of an adult cat kidney. FIG. 11is an enlarged tissue photograph (immunohistochemical staining (×200))of the site shown in FIG. 10. FIG. 12 shows the immunohistochemicalstaining (×400) patterns when the FITC-labeled antibody was used, andFIG. 13 shows the immune electron microscopy (×60,000) images of theendoplasmic reticulum in tubular epithelial cells.

Example 4

(1) The Cauxin Levels in each μg of the Urinary Protein wereQuantitatively compared in cats at various stages of nephropathy.SDS-PAGE was carried out according to the procedure of Example 1(1)(a).

Quantitative western blotting was carried out in the following manner.Thus, 1 μg of the urinary protein derived from a cat with nephropathywas developed by SDS-PAGE and, then, cauxin was detected by westernblotting, as described in Example 2(2)(a). The cauxin levels werecompared based on the differences in band density (FIG. 14). From FIG.14, it was revealed that the urinary excretion of cauxin and the cauxinlevel in urine per total urinary protein markedly decrease with theadvancement in disease stage of nephropathy.

(2) Staining of a Tissue Derived from a Cat with Interstitial Nephritis

A kidney of a cat which had died of nephropathy was fixed with 4%paraformaldehyde and, then, a paraffin-embedded section was prepared andstained with hematoxylin and eosin. It was observed that kidney tubuleshad been broken and become hollow and other cells had entered thecavities (FIG. 15). Further, immunostaining comparison was made in thenumber of cauxin-producing cells in the corticomedullar transitionalzone between a healthy cat and a cat with nephropathy (FIG. 16 and FIG.17). The immunostaining was carried out according to the methoddescribed in Example 3(1)(b). In the cat at the last stage ofnephropathy, the number of cauxin-producing cells was found markedlydecreased.

Example 5

Gold Colloid Immunochromatographic Test Kit

-   (1) A gold colloid-labeled antibody is prepared by conjugating an    appropriate amount of the anti-cauxin antibody to a gold colloid    solution. The preparation of the gold colloid solution and the    labeling of the anti-cauxin antibody were carried out in the    conventional manner.-   (2) A oblong strip-like filer paper is prepared which has a sample    padding site for dropping a liquid sample, a gold colloid phase    where the gold colloid-labeled antibody is positioned, a trapping    site with the anti-cauxin antibody immobilized thereon, and an    absorbent pad for absorbing the reaction mixture, with the four    elements being disposed in line.-   (3) When a cauxin-containing feline urine sample is dropped onto the    sample pad, the sample migrates to the gold colloid phase owing to    the chromatographic phenomenon and urinary cauxin is bound to the    gold colloid-labeled antibody;-   (4) The gold colloid with cauxin bound to the surface thereof then    further diffuses to the trapping site where the gold colloid is    immobilized by the antibody on the trapping site; and-   (5) Upon flocculation of the gold colloid on the trapping site, the    site is found colored red purple. If cauxin is absent in the urine    sample, the gold colloid passes through the trapping site and    migrates to the absorbent pad, so that no coloration can be    observed. When the urinary concentration of cauxin is high, the    amount of the gold colloid flocculate increases, hence the    coloration becomes intense (or the colored area extends). Thus,    quantitative assaying becomes possible.

1. A protein which comprises isolated cauxin, or a salt thereof.
 2. Aprotein or a salt thereof according to claim 1 which is characterized byits serving as a marker for diagnosing feline nephropathy.
 3. A proteinor a salt thereof according to claim 1 or 2 which is characterized by(a) having at least the sequence Asp²⁶ to Pro⁵⁴² out of the amino acidsequence shown under SEQ ID NO: 2 in the sequence listing, or (b) havingthe amino acid sequence shown under SEQ ID NO: 2 in the sequencelisting, or (c) having at least 50% homology with the sequence Asp²⁶ toPro⁵⁴² out of the amino acid sequence shown under SEQ ID NO: 2 in thesequence listing or having at least 50% homology with the amino acidsequence shown under SEQ ID NO: 2 in the sequence listing.
 4. A methodof diagnosing feline nephropathy which comprises quantitatively assayingfor urinary cauxin and detecting feline nephropathy by using as an indexa decrease in the level of cauxin.
 5. A method of diagnosing felinenephropathy according to claim 4 which comprises (i) the step ofsubjecting a urinary protein-containing sample to separation byelectrophoresis, or (ii) the step of assaying for a biological urinarycauxin activity, or (iii) the step of bringing a urinaryprotein-containing sample into contact with an anti-cauxin antibody. 6.A diagnostic agent for feline nephropathy which comprises (i) acauxin-staining agent, or (ii) a reagent for assaying for a biologicalurinary cauxin activity, or (iii) an anti-cauxin antibody.
 7. Acauxin-detecting kit which comprises (a) a sample application site, (b)a labeled antibody-containing site, (c) an antigen detection site and(d) a reaction completion judging site, as disposed in that order on acarrier or support enabling substances to move in a wet condition,wherein (i) the labeled antibody-containing site contains a labeledanti-cauxin antibody which is capable of migrating on the carrier to theantigen detection site and then to the reaction completion judging sitein a wet condition, (ii) an immobilized anti-cauxin antibody is placedon the detection site, and (iii) a site where a second antibodydifferent from the labeled antibody is formed on the reaction completionjudging site, and wherein when a sample is applied to the sampleapplication site, the sample is allowed to migrate on the carrier, elutethe labeled antibody and pass through the immobilized antibody on theantigen detection site and through the second antibody site on thereaction completion judging site, for detecting cauxin in the sample. 8.A cauxin detecting kit for the assay of cauxin in a sample which ischaracterized in that a sample is brought into contact with a substratefor carboxylesterase and the resulting signal is measured.
 9. A cauxindetecting kit characterized in (1) that a sample is brought into contactwith an immobilized anti-cauxin antibody and then with a labeledanti-cauxin antibody or (2) that a sample is brought into contact with alabeled anti-cauxin antibody and then an immobilized anti-cauxinantibody, and cauxin is assayed utilizing the label as an indicator.